Immunocompatible chorionic membrane products

ABSTRACT

Provided herein is a placental product comprising an immunocompatible chorionic membrane. Such placental products can be cryopreserved and contain viable therapeutic cells after thawing. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/030,507 filed on Feb. 18, 2011, which claims priority to:

U.S. Provisional Application Ser. No. 61/338,464 entitled “SelectivelyImmunodepleted Chorionic Membranes”, filed on Feb. 18, 2010,

U.S. Provisional Application Ser. No. 61/338,489 entitled “SelectivelyImmunodepleted Amniotic Membranes”, filed on Feb. 18, 2010, and

U.S. Provisional Application Ser. No, 61/369,562 entitled “TherapeuticProducts Comprising Vitalized Placental Dispersions filed on Jul. 30,2010, the contents of which are hereby incorporated by reference intheir entireties.

U.S. application Ser. No. 13/030,507 was co-filed on Feb. 18, 2011 with,and incorporates by reference, applications entitled:

“Methods of Manufacture of Immunocompatible Chorionic Membrane Products”which received U.S. application Ser. No. 13/030,539,

“Immunocompatible Amniotic Membrane Products” which received U.S.application Ser. No. 13/030,551,

“Methods of Manufacture of Immunocompatible Amniotic Membrane Products”which received U.S. application Ser. No. 13/030,562,

“Therapeutic Products Comprising Vitalized Placental Dispersions” “whichreceived U.S. application Ser. No. 13/030,580, and

“Methods of Manufacture of Therapeutic Products Comprising VitalizedPlacental Dispersions” “which received U.S. application Ser. No.13/030,595, which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present technology relates to products to facilitate wound healingsuch as placenta membrane-derived products and biologic skinsubstitutes. The present technology relates to products to protectinjured or damaged tissue, or as a covering to prevent adhesions, toexclude bacteria, to inhibit bacterial activity, or to promote healingor growth of tissue. The field also relates to methods of manufacturingand methods of use of such membrane-derived products.

BACKGROUND OF THE INVENTION

Fresh or decellularized placental membranes have been used topically insurgical applications since at least 1910 when Johns Hopkins Hospitalreported the use of placental membrane for dermal applications.Subsequently unseparated amnion and chorion were used as skinsubstitutes to treat burned or ulcerated surfaces. During the 1950's and60's Troensegaard-Hansen applied boiled amniotic membranes to chronicleg ulcers.

The human chorionic membrane (CM) is one of the membranes that existsduring pregnancy between the developing fetus and mother. It is formedby extraembryonic mesoderm and the two layers of trophoblast andsurrounds the embryo and other membranes. The chorionic villi emergefrom the chorion, invade the endometrium, and allow transfer ofnutrients from maternal blood to fetal blood.

Both fresh and frozen CMs have been used for wound healing therapy. Whenfresh CM is used, there is increased risk of disease transmission.According to published reports, fresh placental tissue, for example,chorionic tissue exhibits cell viability of 100%, however within 28 daysof storage above 0° C. diminished cell viability to 15 to 35%. Freezingover a time of 3 weeks reduced cell viability to 13 to 18%, regardlessof the temperature or medium. As the CM is believed to be immunogenic,it has not been used in commercial wound healing products.

Two placental tissue graft products containing living cells, Apligrafand Dermagraft, are currently commercially available. Both Apligraf andDermagraft comprise ex vivo cultured cells. Neither Apligraf norDermagraft comprise stem cells. Furthermore, neither Apligraf norDermagraft comprise Insulin-like Growth Factor Binding Protein-1(IGFBP-1) and adiponectin, which are key factors in the natural woundhealing process. In addition, neither Apligraf nor Dermagraft exhibit aprotease-to-protease inhibitor ratio favorable for wound healing. Aswound healing is a multi-factorial biological process, many factors areneeded to properly treat a wound; products having non-native cellularpopulations are less capable of healing wounds relative to a producthaving an optimal population of cells representing the native array. Itwould represent an advance in the art to provide a chorion-derivedbiologic skin substitute comprising a population of cells representingthe native array of factors, including, for example, growth factors andcytokines.

Apligraf is a living, bi-layered skin substitute manufactured usingneonatal foreskin keratinocytes and fibroblasts with bovine Type Icollagen. As used in this application, Apligraf refers to the productavailable for commercial sale in November 2009.

Dermagraft is cryopreserved human fibroblasts derived from newbornforeskin tissue seeded on extracellular matrix. According to its productliterature, Dermagraft requires three washing steps before use whichlimits the practical implementation of Dermagraft as a skin substituterelative to products that require less than three washing steps. As usedin this application, Dermagraft refers to the product available forcommercial sale in November 2009.

Engineered skin substitutes such as Apligraf and Dermagraft do notprovide the best potential for wound healing because they comprisesub-optimal cellular compositions and therefore do not provide properwound healing. For example, neither Apligraf nor Dermagraft comprisesstem cells and, as a result, the ratio between different factorssecreted by cells does not enable efficient wound healing. Additionally,some factors that are important for wound healing, including EGF,IGFBP1, and adiponectin are absent from both Apligraf and Dermagraft.Additionally, some factors, including MMPs and TIMPs, are present inproportions that differ greatly from the proportions found in thenatural wound healing process; this difference significantly alters,among other things, upstream inflammatory cytokine pathways which inturn allows for sub-optimal micro-environments at the wound site. Thepresent inventors have identified a need for the development ofchorionic membrane products that more closely resemble natural tissue.

Paquet-Fifield et al. report that mesenchymal stem cells and fibroblastsare important for wound healing (J Clin Invest, 2009, 119: 2795). Noproduct has yet been described that comprise mesenchymal stem cells andfibroblasts.

Both MMPs and TIMPs are among the factors that are important for woundhealing. However, expression of these proteins must be highly regulatedand coordinated. Excess of MMPs versus TIMPs is a marker of poor chronicwound healing (Liu et al, Diabetes Care, 2009, 32: 117; Mwaura et al,Eur J Vasc Endovasc Surg, 2006, 31: 306; Trengove et al, Wound Rep Reg,1999, 7: 442; Vaalamo et al, Hum Pathol, 1999, 30: 795).

α2-macroglobulin is known as a plasma protein that inactivatesproteinases from all 4 mechanistic classes: serine proteinases, cysteineproteinases, aspartic proteinases, and metalloproteinases (Borth et al.,FASEB J, 1992, 6: 3345; Baker et al., J Cell Sci, 2002, 115:3719).Another important function of this protein is to serve as a reservoirfor cytokines and growth factors, examples of which include TGF, PDGF,and FGF (Asplin et al, Blood, 2001, 97: 3450; Huang et al, J Biol Chem,1988; 263: 1535). In chronic wounds like diabetic ulcers or venousulcers, the presence of high amount of proteases leads to rapiddegradation of growth factors and delays in wound healing. Thus, aplacental membrane skin substitute comprising α2-macroglobulin wouldconstitute an advance in the art.

bFGF modulates a variety of cellular processes including angiogenesis,tissue repair, and wound healing (Presta et al., 2005, Reuss et al.,2003, and Su et al., 2008). In wound healing models, bFGF has been shownto increase wound closure and enhance vessel formation at the site ofthe wound (Greenhalgh et al., 1990).

An in vitro cell migration assay is important for assessing the woundhealing potential of a skin substitute. The process of wound healing ishighly complex and involves a series of structured events controlled bygrowth factors (Goldman, Adv Skin Wound Care, 2004, 1:24). These eventsinclude increased vascularization, infiltration by inflammatory immunecells, and increases in cell proliferation. The beginning stages ofwound healing revolve around the ability of individual cells to polarizetowards the wound and migrate into the wounded area in order to closethe wound area and rebuild the surrounding tissue. Keratinocytes are theprimary cell type of the epithelial layer. Upon proper stimulation, theyare implicated in the wound healing process (Pastar et al, 2008 andBannasch et al., 2000). Specifically, they proliferate and migrate intothe wound area to promote healing. An assay capable of evaluating thewound healing potential of skin substitutes by examining the correlationbetween cell migration and wound healing would represent an advance inthe art.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutically acceptable placentalproduct.

A placental product according to the present invention comprises animmunocompatible chorionic membrane in a cryopreservation medium(optionally cryopreserved) and viable native therapeutic cells andnative therapeutic factors.

In some embodiments, the placental product further comprises an amnioticmembrane that is selectively devitalized.

There is now provided a placental product that is selectively depletedof substantially all immunogenic cells.

There is now provided a placental product that does not contain ex vivocultured cells.

There is now provided a placental product that comprises at least one ofEpidermal Growth Factor, IGFBP1, and Adiponectin.

Optionally, the therapeutic factors include one or more of IGFBP1,adiponectin, α2-macroglobulin, bFGF, and EGF. Optionally, thetherapeutic factors include MMP-9 and TIMP1, wherein the ratio ofMMP-9:TIMP1 is from about 7 to about 10. Optionally, the therapeuticfactors include IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9,and TIMP1. Optionally, the therapeutic factors include IGFBP1,adiponectin, α2-macroglobulin, bFGF, MMP-9, and TIMP1, wherein the ratioof MMP-9:TIMP1 is from about 7 to about 10. Optionally, the therapeuticfactor is present in a substantial amount in comparison to theequivalent unprocessed human placental membrane. Optionally, eachplacental product embodiment optionally is devoid of ex-vivo expandedcultured cells.

The present invention also provides a method of manufacturing aplacental product comprising: obtaining a placenta, wherein the placentacomprises a chorionic membrane, selectively depleting the placenta ofimmunogenicity, and cryopreserving the placenta, thereby providing aplacental product. According to the present invention, the selectivedepletion step comprises removing immunogenic cells (e.g. CD14+macrophages and/or trophoblasts) and/or immunogenic factors (e.g. TNFα).Optionally, the selective depletion step comprises selectivelyimmunodepleting the placenta, whereby the placental product is purifiedfrom immunogenic cells and/or immunogenic factors. Optionally, theselective depletion step comprises removing a layer of trophoblasts, forexample, by treatment with a digestive enzyme and/or mechanical removal.Optionally, the selective depletion step comprises removing CD14+macrophages by a cryoprocess wherein the placental product is incubatedfor a period of time (e.g. about 30-60 mins.) at a temperature abovefreezing (e.g. at 2-8° C.), and then freezing, whereby CD14+ macrophagesare selectively killed relative to therapeutic cells.

The present invention also provides a method of screening a placentalproduct for therapy comprising assaying the placental product forimmunogenicity and/or therapeutic value. Optionally, the step ofassaying the placental product for immunogenicity comprises a MixedLymphocyte Reaction (MLR) and/or Lipopolysaccharide (LPS)-induced TumorNecrosis Factor (TNF)-α secretion. Optionally, the step of assaying theplacental product for therapeutic value comprises assaying the placentalproduct for cell migration induction.

The present invention also provides a method of treating a subjectcomprising administering a placental product to the subject. Optionally,the step of administering comprises applying the placental product to awound, for example, topically applying the placental product to a skinwound. In one embodiment, a placental product is used in a tendon orligament surgery to promote healing of a tendon or ligament.

The present inventors have identified a need for the development ofchorionic membrane products comprising at least one of IGFBP1, andadiponectin, providing superior wound healing properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-H depict freezing rates of various freezing methods of themembrane products either in a styrofoam box (1A, 1B, 1E, and 1F) or on afreezer shelf (1C, 1D, 1G and 1H) under various cryopreservationconditions.

FIG. 2 depicts process cell recovery as a function of cryo volume.

FIG. 3 depicts process cell recovery as a function of refrigerationtime.

FIG. 4A-F show representative images of the live/dead staining of theepithelial layer of fresh amniotic membrane (A); epithelial layer ofcryopreserved amniotic membrane (B); stromal layer of fresh amnioticmembrane (C); stromal layer of cryopreserved amniotic membrane (D);fresh chorionic membrane (E); and cryopreserved chorionic membrane (F).

FIG. 5 depicts IL-2sR concentrations of various manufacturingintermediates.

FIG. 6 depicts IL-2sR concentrations of various manufacturingintermediates.

FIG. 7A-C depict TNF a concentrations from LPS-induced secretion byplacental tissues.

FIG. 8A-C show representative images of the live/dead staining of theepithelial layer of fresh amniotic membrane. FIG. 8-A shows arepresentative image of passage 2 cells, FIG. 8-B shows a representativeimage of MSCs isolated and expanded from human bone marrow aspirate andFIG. 8-C shows a representative image of passage 2 cells stainingpositively for alkaline phosphatase.

FIG. 9 depicts a correlation between IL-2sR release and the number ofCD45+ cells.

FIG. 10 depicts a correlation between the amount of CD45+ cells presentin amnion-derived cell suspensions and immunogenicity in MLR in vitro.

FIG. 11A-C depict expression of EGF (A), IGFBP1 (B), and Adiponectin (C)in amniotic and/or chorionic membranes.

FIG. 12A-B depict expression of IFN-2α (FIG. 12A) and TGF-β3 (FIG. 12B)in amniotic membrane homogenates.

FIG. 13A-B depict expression of BMP-2, BMP-7, PLAB, PLGF (A), and IGF-1(B) in chorionic membrane homogenates.

FIG. 14 depicts the ratio of MMPs to TIMPs in various membrane products.

FIG. 15 depicts bFGF levels in amniotic and chorionic membranes (CM).

FIG. 16 depicts representative expression of bFGF in chorionic tissuesamples derived from two separate placenta donors.

FIG. 17 depicts a schematic of the cell migration assay.

FIG. 18 depicts the results of cell migration assay of various membranepreparations.

FIG. 19A-C depict growth factor and adiponectin expression in proteinextracts of various membrane preparations; FIG. 19A depicts EGFexpression, FIG. 19B depicts IGFBP1 expression; and FIG. 19C depictsadiponectin expression.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following definitions apply:

“Examplary” (or “e.g.” or “by example”) means a non-limiting example.

“hCMSCs: means human chorionic membrane stromal cells. hCMSCs aregenerally positive for CD73, CD70, CD90, CD105, and CD166; negative forCD45 and CD34, hCMSCs differentiate to mesodermal lineages (osteogenic,chondrogenic, and adipogenic).

“Selective depletion of immunogenicity” or “selective depletion ofimmunogenic cells or factors” or “selective depletion” means a placentalproduct that retains live therapeutic cells and/or retains therapeuticefficacy for the treatment of tissue injury yet is free, substantiallyfree, or depleted of at least one of immune cell type (e.g. CD14+macrophages, trophoblasts, and/or vascular-tissue derived cells) and/orimmunogenic factor that are otherwise present in a native placenta orchorionic membrane.

“MSC” means mesenchymal stem cells and include fetal, neonatal, adult,or post-natal. “MSCs” include chorionic MSCs (CMSCs). MSCs generallyexpress one or more of CD73, CD90, CD105, and CD166.

“Native cells” means cells that are native, resident, or endogenous tothe placental membrane, i.e. cells that are not exogenously added to theplacental membrane.

“Native factors” means placental membrane factors that are native,resident, or endogenous to the placental membrane, i.e. factors that arenot exogenously added to the placental membrane.

“Placental products” means the instant placental products disclosedherein.

“Substantially free” means present in only a negligible amount or notpresent at all. For example, when a cell is abundant at least than about20% or less than about 10% or less than about 1% of the amount in anunprocessed sample.

“Substantial amount” of an element of the present invention, e.g. nativefactors, therapeutic factors, or selective depletion, means a value atleast about 2% or at least 10% in comparison to an unprocessed, notcryopreserved, fresh membrane sample. A substantial amount canoptionally be at least about 50%.

“Therapeutic cells” or “beneficial cells” means stromal cells, MSCs,and/or fibroblasts.

“Therapeutic factors” means placenta- or chorionic membrane-derivedfactors that promote wound healing. Examples include IGFBP1,adiponectin, α2-macroglobulin, and/or bFGF. Other examples include MMP-9and TIMP1.

“Stromal cells” refers to a mixed population of cells present(optionally in native proportions) composed of neonatal mesenchymal stemcells and neonatal fibroblasts. Both neonatal mesenchymal stem cells andneonatal fibroblasts are immunoprivileged; neither express surfaceproteins present on immunogenic cell types.

In some embodiments, the present technology discloses placental productsfor clinical use, including use in wound healing such as diabetic footulcers, venous leg ulcers, and burns. The manufacturing processoptionally eliminates essentially all potentially immunogenic cells fromthe placental membrane while preserving of specific cells that play animportant role in wound healing.

In some embodiments, the present technology discloses a placentalproduct that is selectively devitalized. There is now provided aplacental product that is selectively depleted of substantially allimmunogenic cells. There is now provided a placental product that doesnot contain ex vivo cultured cells. There is now provided a placentalproduct that comprises at least one of IGFBP1, and adiponectin. There isnow provided a placental product that comprises IGFBP1. There is nowprovided a placental product that comprises adiponectin.

In some embodiments, the present technology discloses a method ofcyropreserving a placental product that preserves the viability ofspecific beneficial cells that are the primary source of factors for thepromotion of healing to the wound healing process while selectivelydepleting immunogenic cells (e.g. killing or rendering non-immunogenic)from the chorionic membranes.

In some embodiments, the present technology discloses a bioassay to testimmunogenicity of manufactured placental products.

In some embodiments, the present technology discloses a placentalproduct exhibiting a ratio of MMP:TIMP comparable to that exhibited invivo. The present inventors have identified a need for the developmentof placental products exhibiting a ratio of MMP-9 and TIMP1 of about7-10 to one.

In some embodiments, the present technology discloses a placentalproduct that comprises α2-macroglobulin.

The present inventors have identified a need for the development ofplacental products that comprise α2-macroglobulin.

There is now provided a placental product that inactivates substantiallyall serine proteinases, cysteine proteinases, aspartic proteinases, andmetalloproteinases present in the chorionic membrane. There is nowprovided a placental product that inactivates substantially all serineproteinases present in the chorionic membrane. There is now provided aplacental product that inactivates substantially all cysteineproteinases present in the chorionic membrane. There is now provided aplacental product that inactivates substantially all asparticproteinases present in the chorionic membrane. There is now provided aplacental product that inactivates substantially all metalloproteinasespresent in the chorionic membrane.

In some embodiments, the present technology discloses a placentalproduct that comprises bFGF, optionally in a substantial amount.

In some embodiments, the present technology discloses a placentalproduct exhibiting a protease-to-protease inhibitor ratio favorable forwound healing, optionally in a substantial amount.

In some embodiments, the present technology discloses a cell migrationassay capable of evaluating the wound-healing potential of a placentalproduct.

IGFBP1 and adiponectin are among the factors that are important forwound healing. Evaluation of proteins derived from placental productsprepared according to the presently disclosed technology reveal thatbFGF is one of the major factors secreted in substantial higherquantities by the chorionic membrane. Additionally, the importance ofEGF for wound healing together with high levels of bFGF detected in thepresently disclosed chorionic membranes support selection of bFGF as apotency marker for evaluation of membrane products manufactured forclinical use pursuant to the present disclosure.

The present technology discloses a cryopreservation procedure for aplacental products that selectively depletes immunogenic cells from thea chorionic membranes and preserves the viability of other beneficialcells (including at least one of mesenchymal stem cells, and fibroblastsin some embodiments and all of mesenchymal stem cells and fibroblasts insome embodiments) that are the primary source of factors for thepromotion of healing. During the development of cryopreservationmethodology for chorionic membranes, the inventors of the presentapplication evaluated key parameters of cryopreservation includingvolume of cryopreservative solution, effect of tissue equilibrationprior to freezing, and cooling rates for a freezing procedures.

Placental products, their usefulness, and their immunocompatability aresurprisingly enhanced by depletion of maternal trophoblast and selectiveelimination of CD14+ fetal macrophages. Immunocompatability can bedemonstrated by any means commonly known by the skilled artisan, suchdemonstration can be performed by the mixed Lymphocyte Reaction (MLR)and by lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-αsecretion.

The instant placental products contain bFGF, optionally at a substantialconcentration.

The instant placental products optionally secrete factors that stimulatecell migration and/or wound healing. The presence of such factors can bedemonstrated by any commonly recognized method. Optionally, the factorsare in a substantial amount.

For example, commercially available wound healing assays exist (CellBiolabs) and cell migration can be assessed by cell line (HMVEC, LonzaInc.). In one embodiment, conditioned medium from the present placentalproducts enhance cell migration.

The placental products disclosed herein are useful in treating a numberof wounds including: tendon repair, cartilage repair (e.g. femoralcondyle, tibial plateau), ACL replacement at the tunnel/bone interface,dental tissue augmentation, fistulas (e.g. Crohn's disease, G-tube,tracheoesophogeal), missing tissue at adhesion barriers (e.g. nasalseptum repair, vaginal wall repair, abdominal wall repair, tumorresection), dermal wounds (e.g. partial thickness burns, toxic epidermalnecrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g.diabetic ulcers (e.g. foot), venous leg ulcers), surgical wounds, herniarepair, tendon repair, bladder repair, periosteum replacement, keloids,organ lacerations, epithelial defects, and repair or replacement of atympanic membrane.

The placental products disclosed herein exhibit one or more of thefollowing properties beneficial to the wound healing process:

-   -   a. approximate number of cells per cm² being about 20,000 to        about 200,000,    -   b. thickness of about 40 to about 400 μm,    -   c. a thin basement membrane,    -   d. low immunogenicity,    -   e. cryopreserved/cryopreserveable, and    -   f. human Chorionic Membrane Stromal Cells (hCMSC).

The present inventors have now identified a need for the development ofplacental products that do not contain ex vivo cultured cells.

The present inventors have now identified a need for the development ofplacental products comprising IGFBP1.

The present inventors have now identified a need for the development ofplacental products comprising adiponectin.

The present inventors have now identified a need for the development ofplacental products exhibiting a protease-to-protease inhibitor ratiofavorable for wound healing.

The present inventors have now identified a need for the development ofa method of cyropreserving placental products that preserves theviability of specific cells that are other beneficial cells that are theprimary source of factors for the promotion of healing to the woundhealing process while selectively depleting immunogenic cells fromchorionic membranes.

The present inventors have now identified a need for the development ofa bioassay to test immunogenicity of manufactured placental products.

The present inventors have now identified a need for the development ofplacental products exhibiting a ratio of MMP to TIMP comparable to thatexhibited in vivo. The present inventors have now identified a need forthe development of placental products exhibiting a ratio of MMP-9 andTIMP1 of about 7-10 to one.

The present inventors have now identified a need for the development ofplacental products that comprise α2-macroglobulin.

The present inventors have now identified a need for the development ofplacental products that inactivate serine proteinases, cysteineproteinases, aspartic proteinases, and metalloproteinases. The presentinventors have now identified a need for the development of placentalproducts that inactivate serine proteinases. The present inventors havenow identified a need for the development of placental products thatinactivate cysteine proteinases. The present inventors have nowidentified a need for the development of placental products thatinactivate aspartic proteinases. The present inventors have nowidentified a need for the development of placental products thatinactivate metalloproteinases.

The present inventors have now identified a need for the development ofplacental products that comprise bFGF.

The present inventors have now identified a need for the development ofa cell migration assay to evaluate the potential of placental membraneproducts.

The present inventors have now identified a need for the development ofa placental product for wound healing that comprises mesenchymal stemcells and fibroblasts.

Placental Product

Overview

One embodiment of the present invention provides a placental productcomprising a cryopreservation medium and a chorionic membrane, whereinthe chorionic membrane comprises viable therapeutic native cells andnative therapeutic factors, and wherein the cryopreservation mediumcomprises a cryopreserving amount of a cryopreservative. According tothis embodiment, the chorionic membrane is substantially free of atleast one at least one or 2 or 3 immunogenic cell types such as:trophoblasts, CD14+ macrophages, and vascularized tissue-derived cells.

In one embodiment, the chorionic membrane comprises one or more layerswhich exhibit the architecture of the native chorionic membrane (e.g.has not been homogenized or treated with collagenase).

In one embodiment, the placental product is suitable for dermalapplication to a wound.

With the teachings provided herein, the skilled artisan can now producethe present placental products. The present disclosure provides methodsof manufacture that produce the technical features of the presentplacental products. Accordingly, in one embodiment, the placentalproduct is manufactured by steps taught herein. The present placentalproducts are not limited to products manufactured by the methods taughthere. For example, products of the present invention could be producedthrough methods that rely on screening steps; e.g. steps to screen forpreparations with the described technical features and superiorproperties.

The present placental products comprises one or more of the followingtechnical features:

-   -   a. the viable therapeutic native cells are capable of        differentiating into cells of more than one lineage (e.g.        osteogenic, adipogenic and/or chonodrogenic lineages),    -   b. the native therapeutic factors include IGFBP1, optionally        present in a substantial amount,    -   c. the native therapeutic factors include adiponectin,        optionally present in a substantial amount,    -   d. the native therapeutic factors include α2-macroglobulin,        optionally present in a substantial amount,    -   e. the native therapeutic factors include bFGF, optionally        present in a substantial amount,    -   f. the native therapeutic factors include EGF, optionally        present in a substantial amount,    -   g. the native therapeutic factors include MMP-9 and TIMP1,        optionally present in a substantial amount,    -   h. the native therapeutic factors include MMP-9 and TIMP1 in a        ratio of about 7 to about 10,    -   i. the placental product does not comprise ex-vivo cultured        cells,    -   j. the cryopreservative medium is present in an amount of        greater than about 20 ml or greater than about 50 ml,    -   k. the cryopreservative comprises DMSO,    -   l. cryopreservative comprises DMSO in a majority amount,    -   m. the cryopreservation medium further comprises albumin,        optionally wherein the albumin is HSA,    -   n. the cryopreservative comprises DMSO and albumin (e.g. HSA),    -   o. the chorionic membrane comprises about 5,000 to about 240,000        cells/cm2 or about 20,000 to about 60,000 cells/cm2,    -   p. the chorionic membrane comprises 20,000 to about 200,000        cells/cm2, with a cell viability of at least about 70%,    -   q. comprises at least: about 7,400 or about 15,000 or about        23,217, or about 35,000, or about 40,000 or about 47,800 of        stromal cells per cm2 of the chorionic membrane,    -   r. comprises about 5,000 to about 50,000 of stromal cells per        cm2 of the chorionic membrane,    -   s. comprises about 4% to about 46% of viable non-culturally        expanded fibroblasts per cm2 of the placental product,    -   t. comprises stromal cells wherein at least: about 40%, or about        50%, or about 60%, or about 70%, or about 74.3%, or about 83.4        or about 90%, or about 92.5% of the stromal cells are viable        after a freeze-thaw cycle,    -   u. comprises stromal cells wherein about 40% to about 92.5% of        the stromal cells are viable after a freeze-thaw cycle,    -   v. the chorionic membrane has a thickness of about 40 μm to        about 400 μm,    -   w. secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280        pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2        cm piece of the tissue product in a tissue culture medium and        exposing the tissue product to a bacterial lipopolysaccharide        for about 20 to about 24 hours,    -   x. cryopreservation and thawing, secretes less than about any        of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue        culture medium upon placing a 2 cm×2 cm piece of the tissue        product in a tissue culture medium and exposing the tissue        product to a bacterial lipopolysaccharide for about 20 to about        24 hours,    -   y. after refrigeration, cryopreservation and thawing, secretes        less than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α        into a tissue culture medium upon placing a 2 cm×2 cm piece of        the tissue product in a tissue culture medium and exposing the        tissue product to a bacterial lipopolysaccharide for about 20 to        about 24 hours,    -   z. the maternal side of the chorionic membrane comprises        fragments of extracellular matrix proteins in a concentration        substantially greater than that of a native, unprocessed        chorion, optionally wherein the chorionic membrane has been        treated with Dispase II or wherein a substantial portion of the        protein fragments comprises terminal leucine or phenylalanine,    -   aa. further comprises an amniotic membrane,    -   bb. further comprises an amniotic membrane, wherein the amniotic        membrane comprises a layer of amniotic epithelial cells,    -   cc. further comprises an amniotic membrane, wherein the amniotic        membrane and the chorionic membrane are associated to one        another in the native configuration,    -   dd. further comprises an amniotic membrane, wherein the amniotic        membrane and the chorionic membrane are not attached to one        another in the native configuration,    -   ee. further comprises an amniotic membrane wherein the chorionic        membrane comprises about 2 to about 4 times more stromal cells        relative to the amniotic membrane,    -   ff. does not comprise an amniotic membrane,    -   gg. the chorionic membrane comprises about 2 to about 4 times        more stromal cells relative to an amniotic membrane of the same        area derived from the same placenta, and    -   hh. is suitable for dermal application to a wound.

Cells

According to the present invention, a placental product comprises nativetherapeutic cells of the chorionic membrane. The cells comprise one ormore of stromal cells, MSCs, and fibroblasts.

In one embodiment, the native therapeutic cells comprise viable stromalcells.

In one embodiment, the native therapeutic cells comprise viable MSCs.

In one embodiment, the native therapeutic cells comprise viablefibroblasts.

In one embodiment, the native therapeutic cells comprise viable MSCs andviable fibroblasts.

In one embodiment, the native therapeutic cells comprise viable MSCs andviable fibroblasts.

In one embodiment, the native therapeutic cells comprise viable stromalcells and viable epithelial cells.

In one embodiment, the therapeutic native cells are viable cells capableof differentiating into cells of more than one lineage (e.g. osteogenic,adipogenic and/or chonodrogenic lineages).

In one embodiment, the chorionic membrane comprises about 10,000 toabout 360,000 cells/cm² or about 40,000 to about 90,000 cells/cm².

In one embodiment, the chorionic membrane comprises at least: about7,400 or about 15,000 or about 23,217, or about 35,000, or about 40,000or about 47,800 of stromal cells per cm² of the chorionic membrane.

In one embodiment, the chorionic membrane comprises about 5,000 to about50,000 of stromal cells per cm² of the chorionic membrane.

In one embodiment, the chorionic membrane comprises stromal cellswherein at least: about 40%, or about 50%, or about 60%, or about 70%,or about 74.3%, or about 83.4 or about 90%, or about 92.5% of thestromal cells are viable after a freeze-thaw cycle.

In one embodiment, the chorionic membrane comprises stromal cellswherein about 40% to about 92.5% of the stromal cells are viable after afreeze-thaw cycle.

In one embodiment, the chorionic membrane (of the placental product)comprises fibroblasts in about 50% to about 90% of the total cells.

In one embodiment, the chorionic membrane comprises CD14+ macrophage inan amount of less than about 5% or less than about 1% or less than about0.5%, optionally as demonstrated by a substantial decrease in LPSstimulation of TNFα release.

In one embodiment, the placental product comprises about 2 to about 4times more stromal cells relative to an amniotic membrane of the samearea derived from the same placenta.

In one embodiment, the placental product further comprises an amnioticmembrane, wherein the placental product contains about 2 to about 4times more stromal cells relative to the amniotic membrane.

In one embodiment, the placental product further comprises an amnioticmembrane, wherein the amniotic membrane comprises a layer of amnioticepithelial cells.

In one embodiment, the placental product is substantially free oftrophoblasts.

In one embodiment, the placental product is substantially free offunctional CD14+ macrophages.

In one embodiment, the placental product is substantially free ofvascularized tissue-derived cells.

In one embodiment, the placental product is substantially free oftrophoblasts, functional CD14+ macrophages, and vascularizedtissue-derived cells. Optionally, the placental product comprises viablestromal cells. Optionally, the placental product comprises viable MSCs.Optionally, the placental product comprises viable fibroblasts.Optionally, the placental product comprises viable MSCs and viablefibroblasts.

In one embodiment, the placental product is substantially free ofmaternal decidual cells.

In one embodiment, the placental product is substantially free ofmaternal dendritic cells, leukocytes and/or trophoblast cells.

In one embodiment, the chorionic membrane (of the placental product)comprises MSCs in an amount of about 5% to about 30%, about 5% to about25%, about 5% to about 20%, about 5% to about 15%, about 3% to about12%, at least about 5%, at least about 10%, or at least about 15%,relative to the total number of cells in the chorionic membrane.Optionally, at least: about 40%, about 50%, about 60%, or about 70% ofthe MSCs are viable after a freeze-thaw cycle.

In one embodiment, the chorionic membrane (of the placental product)comprises fibroblasts in an amount of about 50% to about 95%, about 60%to about 90%, or about 70% to about 85%, relative to the total number ofcells in the chorionic membrane. Optionally, at least: about 40%, about50%, about 60%, or about 70% of the fibroblasts are viable after afreeze-thaw cycle.

In one embodiment, the chorionic membrane (of the placental product)comprises functional macrophages in an amount of less than about any of:5%, 4%, 3%, 2%, 1%, or 0.1%.

In one embodiment, the chorionic membrane (of the placental product)comprises MSCs and functional macrophages in a ratio of greater thanabout any of: 3:1, 4:1, 5:1, 7:1, 10:1, 12:1, or 15:1.

In one embodiment, the chorionic membrane comprises fibroblasts andfunctional macrophages in a ratio of greater than about any of: 14:1,15:1, 16:1, 17:1, 28:1, 30:1, 35:1, 45:1, or 50:1.

In one embodiment, the chorionic membrane (of the placental product)comprises fibroblasts and MSCs in a ratio of: about 9:2 to about 17:3.

In one embodiment, the chorionic membrane (of the placental product)comprises MSCs in an amount of at least about 1,500 cells/cm², at leastabout 3,000 cells/cm², about 15,000 to about 9,000 cells/cm², or about3,000 to about 9,000 cells/cm². Optionally, at least: about 40%, about50%, about 60%, or about 70% of the MSCs are viable after a freeze-thawcycle.

In one embodiment, the chorionic membrane (of the placental product)comprises fibroblasts in an amount of at least about 7,000 cells/cm², atleast about 14,000 cells/cm², about 7,000 to about 51,000 cells/cm², orabout 14,000 to about 51,000 cells/cm². Optionally, at least: about 40%,about 50%, about 60%, or about 70% of the fibroblasts are viable after afreeze-thaw cycle.

In one embodiment, the chorionic membrane (of the placental product)comprises functional macrophages in an amount of less than about 3,000cells/cm², or less than about 1,000 cells/cm².

In one embodiment, the placental product is substantially free ofex-vivo cultured cells.

Placental Factors

According to the present invention, a placental product comprises nativetherapeutic factors of the chorionic membrane.

In one embodiment, the factors include one or more of: IGFBP1,adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally,the factors are present in amounts/cm² that are substantially similar tothat of a native chorionic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include IGFBP1, adiponectin,α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally, the factorsare present in ratios that are substantially similar to that of a nativechorionic membrane or layer thereof. Optionally, the factors are presentin amounts/cm² that are substantially similar to that of a nativechorionic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include MMP-9 and TIMP1 in a ratio ofabout 7 to about 10 (e.g. about 7). Optionally, the factors are presentin amounts/cm² that are substantially similar to that of a nativechorionic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include one or more (e.g. a majority orall) of the factors listed in Table 15. Optionally, the factors arepresent in ratios that are substantially similar to that of a nativechorionic membrane or layer thereof.

Optionally, the factors are present in amounts/cm² that aresubstantially similar to that of a native chorionic membrane or layerthereof (e.g. ±10% or 20%).

In one embodiment, the placental product secretes substantially lessTNF-α/cm² than a native, unprocessed chorionic membrane.

In one embodiment, the placental product secretes substantially lessTNF-α/cm² than a native placental product upon stimulation by LPS or CT.

In one embodiment, the placental product secretes less than about anyof: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culturemedium upon placing a 2 cm×2 cm piece of the tissue product in a tissueculture medium and exposing the tissue product to a bacteriallipopolysaccharide for about 20 to about 24 hours.

In one embodiment, after cryopreservation and thawing, the placentalproduct secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm pieceof the tissue product in a tissue culture medium and exposing the tissueproduct to a bacterial lipopolysaccharide for about 20 to about 24hours.

In one embodiment, after refrigeration, cryopreservation and thawing,the placental product secretes less than about any of: 420 pg/mL, 350pg/mL, or 280 pg/mL TNF-α into a tissue culture medium upon placing a 2cm×2 cm piece of the tissue product in a tissue culture medium andexposing the tissue product to a bacterial lipopolysaccharide for about20 to about 24 hours.

In one embodiment, the placental product further comprises anexogenously added inhibitor of TNF-α. Optionally, the inhibitor of TNF-αis IL-10.

In one embodiment, the product has been treated with an antibiotic

Architecture

A placental product of the present invention comprises one or morenon-trophoblast layers which exhibit the architecture of the nativechorionic membrane. With the teachings provided herein, the skilledartisan will recognize placental layers that exhibit nativearchitecture, for example, layers that have not been homogenized ortreated with collagenase or other enzyme that substantially disrupts thelayer.

In one embodiment, the placental product comprises a stromal layer withnative architecture.

In one embodiment, the placental product comprises a basement membranewith native architecture.

In one embodiment, the placental product comprises a reticular layerwith native architecture.

In one embodiment, the placental product comprises a reticular layer anda basement layer with native architecture.

In one embodiment, the placental product comprises a stromal layer, abasement layer, and a reticular layer with native architecture.

In one embodiment, the placental product is substantially free oftrophoblasts.

In one embodiment, the placental product comprises a basement membranewith native architecture and the chorionic membrane is substantiallyfree of trophoblasts. Optionally, the maternal side of the placentalproduct comprises fragments of extracellular matrix proteins in aconcentration substantially greater than that of a native chorionicmembrane. Optionally, the placental product has been treated withDispase (e.g. Dispase II) and/or a substantial portion of theextracellular matrix protein fragments comprises terminal leucine orphenylalanine.

In one embodiment, the placental product has a thickness of about 40 μmto about 400 μm.

In one embodiment, the placental product further comprises an amnioticmembrane. Optionally, the amniotic membrane and the chorionic membranein the placental product are associated to one another in the nativeconfiguration. Alternatively, the amniotic membrane and the chorionicmembrane are not attached to one another in the native configuration.

In one embodiment, the placental product does not comprise an amnioticmembrane.

Formulation

According to the present invention, the placental product can beformulated with a cryopreservation medium.

In one embodiment, the cryopreservation medium comprising one or morecell-permeating cryopreservatives, one or more non cell-permeatingcryopreservatives, or a combination thereof.

Optionally, the cryopreservation medium comprises one or morecell-permeating cryopreservatives selected from DMSO, a glycerol, aglycol, a propylene glycol, an ethylene glycol, or a combinationthereof.

Optionally, the cryopreservation medium comprises one or more noncell-permeating cryopreservatives selected from polyvinylpyrrolidone, ahydroxyethyl starch, a polysaccharide, a monosaccharides, a sugaralcohol, an alginate, a trehalose, a raffinose, a dextran, or acombination thereof.

Other examples of useful cryopreservatives are described in“Cryopreservation” (BioFiles Volume 5 Number 4—Sigma-Aldrich®datasheet).

In one embodiment, the cryopreservation medium comprises acell-permeating cryopreservative, wherein the majority of thecell-permeating cryopreservative is DMSO. Optionally, thecryopreservation medium does not comprise a substantial amount ofglycerol.

In one embodiment, the cryopreservation medium comprises DMSO.Optionally, the cryopreservation medium does not comprise glycerol in amajority amount. Optionally, the cryopreservation medium does notcomprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 15%albumin by weight and about 5% to about 20% cryopreservative by volume(e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g.in a majority amount).

In one embodiment, the placental product is formulated in greater thanabout 20 ml or greater than about 50 ml of cryopreservation medium.Optionally, the cryopreservative comprises DMSO (e.g. in a majorityamount). Optionally, the cryopreservation medium does not comprise asubstantial amount of glycerol.

In one embodiment, the placental product is placed on nitrocellulosepaper.

In one embodiment, the placenta is cut into a plurality of sections.Optionally, the sections are less than about 10 cm×10 cm. Optionally,the sections are between about 2 cm×2 cm and 5 cm×5 cm.

Manufacture

Overview

A placental product of the present invention can manufactured from aplacenta in any suitable manner that provides the technical featurestaught herein. According to the present invention, a placental productcomprises at least an immunocompatible chorionic membrane.

In one embodiment, a placental product is manufactured by a methodcomprising:

-   -   a. obtaining a placenta,    -   b. selectively depleting the placenta of immunogenicity; and    -   c. cryopreserving the placenta.

In one embodiment, a placental product is manufactured by a methodcomprising:

-   -   a. obtaining a placenta;    -   b. removing a substantial portion of trophoblasts from the        placenta; and    -   c. cryopreserving the placenta.

Optionally, the method comprises a step of removing the amnioticmembrane or portion thereof (‘an amniotic membrane’) from the placenta.Optionally, the method comprises a step of removing an amniotic membranefrom the placenta without removing a substantial portion of amnioticepithelial cells from the placenta.

Optionally, the step of removing a substantial portion of trophoblastsfrom the placenta comprises treating the placenta with a digestiveenzyme such as a protease (e.g. dispase or dispase II), mechanicallyremoving trophoblasts from the placenta (e.g. by scraping), or acombination thereof.

Optionally, the method comprises a step of removing vascularized tissuefrom the placenta, for example, by lysing red blood cells, by removingblood clots, or a combination thereof.

Optionally, the method comprises a step of treating the placenta withone or more antibiotics.

Optionally, the method comprises a step of selective depletion of CD14+macrophages.

Optionally, the step of cryopreserving the placenta comprises freezingthe placenta in a cryopreservation medium which comprises one or morecell-permeating cryopreservatives, one or more non cell-permeatingcryopreservatives, or a combination thereof.

Optionally, the step of cryopreserving the placenta comprisesrefrigerating for a period of time and then freezing, therebyselectively depleting CD14+ macrophages.

An examplary placental product of the present invention can bemanufactured or provided with a bandage or skin substitute.

Immunocompatability and Selective Depletion

In one embodiment, the invention the placental product isimmunocompatible. Immunocompatability can be accomplished by anyselective depletion step that removes immunogenic cells or factors orimmunogenicity from the placenta (or chorionic membrane thereof).

In one embodiment, the placental product is made immunocompatible byselectively depleting it of functional immunogenic cells. A placenta canbe made immunocompatible by selectively removing immunogenic cells fromthe placenta (or chorionic membrane thereof) relative to therapeuticcells. For example, immunogenic cells can be removed by killing theimmunogenic cells or by purification of the placenta there from.

In one embodiment, the placenta is made immunocompatible by selectivelydepleting trophoblasts, for example, by removal of the trophoblastlayer.

In one embodiment, the placenta is made immunocompatible by selectivedepletion of functional CD14+ macrophages, optionally resulting indepletion of TNFα upon stimulation, or a combination thereof.

In one embodiment, the placenta is made immunocompatible by selectivedepletion of vascularized tissue-derived cells.

In one embodiment, the placenta is made immunocompatible by selectivedepletion of functional CD14+ macrophages, trophoblasts, andvascularized tissue-derived cells.

In one embodiment, the placenta product is made immunocompatible byselective depletion of trophoblasts and/or CD14+ macrophages, optionallyresulting in depletion of TNFα upon stimulation.

Trophoblast Removal

In one embodiment, trophoblasts are depleted or removed from theplacental product. Surprisingly, such a placental product has one ormore of the following superior features:

-   -   a. is substantially non-immunogenic;    -   b. provides remarkable healing time; and    -   c. provides enhanced therapeutic efficacy.

Trophoblasts can be removed in any suitable manner which substantiallydiminishes the trophoblast content of the placental product. Optionally,the trophoblasts are selectively removed or otherwise removed withouteliminating a substantial portion of one or more therapeutic componentsfrom the placenta (e.g. MSCs, placental factors, etc). Optionally, amajority (e.g. substantially all) of the trophoblasts are removed.

One method of removing trophoblasts comprises treating the placenta(e.g. chorion or amnio-chorion) with a digestive enzyme such as dispase(e.g. dispase II) and separating the trophoblasts from the placenta.Optionally, the step of separating comprises mechanical separation suchas peeling or scraping. Optionally, scraping comprises scraping with asoft instrument such as a finger.

One method of removing trophoblasts comprises treating the chorionicmembrane with dispase for about 30 to about 45 minutes separating thetrophoblasts from the placenta. Optionally, the dispase is provided in asolution of about less than about 1% (e.g. about 0.5%). Optionally, thestep of separating comprises mechanical separation such as peeling orscraping. Optionally, scraping comprises scraping with a soft instrumentsuch as a finger.

Useful methods of removing trophoblasts from a placenta (e.g. chorion)are described by Portmann-Lanz et al. (“Placental mesenchymal stem cellsas potential autologous graft for pre- and perinatal neuroregeneration”;American Journal of Obstetrics and Gynecology (2006) 194, 664-73),(“Isolation and characterization of mesenchymal cells from human fetalmembranes”; Journal Of Tissue Engineering And Regenerative Medicine2007; 1: 296-305.), and (Concise Review: Isolation and Characterizationof Cells from Human Term Placenta: Outcome of the First InternationalWorkshop on Placenta Derived Stem Cells”).

In one embodiment, trophoblasts are removed before cryopreservation.

Macrophage Removal

In one embodiment, functional macrophages are depleted or removed fromthe placental product. Surprisingly, such a placental product has one ormore of the following superior features:

-   -   a. is substantially non-immunogenic;    -   b. provides remarkable healing time; and    -   c. provides enhanced therapeutic efficacy.

Functional macrophages can be removed in any suitable manner whichsubstantially diminishes the macrophage content of the placentalproduct. Optionally, the macrophages are selectively removed orotherwise removed without eliminating a substantial portion of one ormore therapeutic components from the placenta (e.g. MSCs, placentalfactors, etc). Optionally, a majority (e.g. substantially all) of themacrophages are removed.

One method of removing immune cells such as macrophages compriseskilling the immune cells by rapid freezing rates such as 60-100° C./min.

Although immune cells can be eliminated by rapid freezing rates, such amethod can also be detrimental to therapeutic cells such as stromalcells (e.g. MSCs). The present inventors have discovered a method ofselectively killing CD14+ macrophages can be selectively killed byrefrigerating the placenta for a period of time (e.g. for at least about10 min such as for about 30-60 mins) at a temperature above freezing(e.g. incubating at 2-8° C.) and then freezing the placenta (e.g.incubating at −80° C.±5° C.). Optionally, the step of freezing comprisesfreezing at a rate of less than 10°/min (e.g. less than about 5°/minsuch as at about 1°/min).

In one embodiment, the step of refrigerating comprises soaking theplacenta in a cryopreservation medium (e.g. containing DMSO) for aperiod of time sufficient to allow the cryopreservation medium topenetrate (e.g. equilibrate with) the placental tissues. Optionally, thestep of freezing comprises reducing the temperature at a rate of about1°/min. Optionally, the step of freezing comprises freezing at a rate ofless than 10°/min (e.g. less than about 5°/min such as at about 1°/min).

In one embodiment, the step of refrigerating comprises soaking theplacenta in a cryopreservation medium (e.g. containing DMSO) at atemperature of about −10-15° C. (e.g. at 2-8° C.) for at least about anyof: 10 min, 20 min, 30 min, 40 min, or 50 min. In another embodiment,the step of refrigerating comprises soaking the placenta in acryopreservation medium (e.g. containing DMSO) at a temperature of about−10-15° C. (e.g. at 2-8° C.) for about any of: 10-120, 20-90 min, or30-60 min. Optionally, the step of freezing comprises freezing at a rateof less than 10°/min (e.g. less than about 5°/min such as at about1°/min).

Removal of Vascularized Tissue-Derived Cells

In one embodiment, vascularized tissue-derived cells (or vasculariedtissue) are depleted or removed from the placental product.Surprisingly, such a placental product has one or more of the followingsuperior features:

-   -   a. is substantially non-immunogenic;    -   b. provides remarkable healing time; and    -   c. provides enhanced therapeutic efficacy.

Vascularized tissue-derived cells can be removed in any suitable mannerwhich substantially diminishes such cell content of the placentalproduct. Optionally, the vascularized tissue-derived cells areselectively removed or otherwise removed without eliminating asubstantial portion of one or more therapeutic components from theplacenta (e.g. MSCs, placental factors, etc).

In one embodiment, removal of vascularized tissue-derived cellscomprises separating the chorion from the placenta by cutting around theplacental skirt on the side opposite of the umbilical cord. The chorionon the umbilical side of the placenta is not removed due to thevascularization on this side.

In one embodiment, removal of vascularized tissue-derived cellscomprises rinsing the chorionic membrane (e.g. with buffer such as PBS)to remove gross blood clots and any excess blood cells.

In one embodiment, removal of vascularized tissue-derived cellscomprises treating the chorionic membrane with an anticoagulant (e.g.citrate dextrose solution).

In one embodiment, removal of vascularized tissue-derived cellscomprises separating the chorion from the placenta by cutting around theplacental skirt on the side opposite of the umbilical cord and rinsingthe chorionic membrane (e.g. with buffer such as PBS) to remove grossblood clots and any excess blood cells.

In one embodiment, removal of vascularized tissue-derived cellscomprises separating the chorion from the placenta by cutting around theplacental skirt on the side opposite of the umbilical cord and treatingthe chorionic membrane with an anticoagulant (e.g. citrate dextrosesolution).

In one embodiment, removal of vascularized tissue-derived cellscomprises separating the chorion from the placenta by cutting around theplacental skirt on the side opposite of the umbilical cord, rinsing thechorionic membrane (e.g. with buffer such as PBS) to remove gross bloodclots and any excess blood cells, and treating the chorionic membranewith an anticoagulant (e.g. citrate dextrose solution).

Selective Depletion of Immunogenicity as Demonstrated by a SubstantialDecrease in LPS Stimulation of TNFα Release.

In one embodiment, the placental product is selectively depleted ofimmunogenicity as demonstrated by a reduction in LPS stimulated TNF-αrelease. depletion d of TNF-α depleted or removed from the placentalproduct.

In one embodiment, TNF-α is depleted by killing or removal ofmacrophages.

In one embodiment, TNF-α is depleted by treatment with an anti-TNF-αantibody.

In one embodiment, TNF-α is functionally depleted by treatment withIL-10, which suppresses TNF-α secretion.

Preservation

A placental product of the present invention may be used fresh or may bepreserved for a period of time. Surprisingly, cryopreservation resultsin immunocompatible placental products.

In one embodiment, a placental product is cryopreserved. A placentalproduct may be cryopreserved by incubation at freezing temperatures(e.g. a −80° C.±5° C.) in a cryopreservative medium.

Cryopreservation can comprise, for example, incubating the placentalproduct at 4° C. for 30-60 min, and then incubating at −80° C. untiluse. The placental product may then be thawed for use. Optionally, theplacental product is cryopreserved in a manner such that cell viabilityis retained surprisingly well after a freeze-thaw cycle.

In one embodiment, cryopreservation comprises storage in acryopreservation medium comprising one or more cell-permeatingcryopreservatives, one or more non cell-permeating cryopreservatives, ora combination thereof. Optionally, the cryopreservation medium comprisesone or more cell-permeating cryopreservatives selected from DMSO, aglycerol, a glycol, a propylene glycol, an ethylene glycol, or acombination thereof. Optionally, the cryopreservation medium comprisesone or more non cell-permeating cryopreservatives selected frompolyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, amonosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose,a dextran, or a combination thereof. Other examples of usefulcryopreservatives are described in “Cryopreservation” (BioFiles Volume 5Number 4—Sigma-Aldrich® datasheet).

In one embodiment, the cryopreservation medium comprises acell-permeating cryopreservative, wherein the majority of thecell-permeating cryopreservative is DMSO. Optionally, thecryopreservation medium does not comprise a substantial amount ofglycerol.

In one embodiment, the cryopreservation medium comprises DMSO.Optionally, the cryopreservation medium does not comprise glycerol in amajority amount. Optionally, the cryopreservation medium does notcomprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 15%albumin by weight and about 5% to about 20% cryopreservative by volume(e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g.in a majority amount).

In one embodiment, cryopreservation comprises placing the placenta onnitrocellulose paper.

In one embodiment, the placenta is cut into a plurality of sectionsbefore cryopreservation. Optionally, the sections are placed onnitrocellulose paper before refrigeration.

Methods of Use

The placental products (e.g. derived from chorionic tissue) of thepresent invention may be used to treat any tissue injury. A method oftreatment may be provided, for example, by administering to a subject inneed thereof, a placental product of the present invention.

A typical administration method of the present invention is topicaladministration. Administering the present invention can optionallyinvolve administration to an internal tissue where access is gained by asurgical procedure.

Placental products can be administered autologously, allogeneically orxenogeneically.

In one embodiment, a present placental product is administered to asubject to treat a wound. Optionally, the wound is a laceration, scrape,thermal or chemical burn, incision, puncture, or wound caused by aprojectile. Optionally, the wound is an epidermal wound, skin wound,chronic wound, acute wound, external wound, internal wounds, congenitalwound, ulcer, or pressure ulcer. Such wounds may be accidental ordeliberate, e.g., wounds caused during or as an adjunct to a surgicalprocedure. Optionally, the wound is closed surgically prior toadministration.

In one embodiment, a present placental product is administered to asubject to treat a burn. Optionally, the burn is a first-degree burn,second-degree burn (partial thickness burns), third degree burn (fullthickness burns), infection of burn wound, infection of excised andunexcised burn wound, loss of epithelium from a previously grafted orhealed burn, or burn wound impetigo.

In one embodiment, a present placental product is administered to asubject to treat an ulcer, for example, a diabetic ulcer (e.g. footulcer).

In one embodiment, a placental product is administered by placing theplacental product directly over the skin of the subject, e.g., on thestratum corneum, on the site of the wound, so that the wound is covered,for example, using an adhesive tape. Additionally or alternatively, theplacental product may be administered as an implant, e.g., as asubcutaneous implant.

In one embodiment, a placental product is administered to the epidermisto reduce rhtids or other features of aging skin. Such treatment is alsousefully combined with so-called cosmetic surgery (e.g. rhinoplasty,rhytidectomy, etc.).

In one embodiment, a placental product is administered to the epidermisto accelerate healing associated with a dermal ablation procedure or adermal abrasion procedure (e.g. including laser ablation, thermalablation, electric ablation, deep dermal ablation, sub-dermal ablation,fractional ablation, and microdermal abrasion).

Other pathologies that may be treated with placental products of thepresent invention include traumatic wounds (e.g. civilian and militarywounds), surgical scars and wounds, spinal fusions, spinal cord injury,avascular necrosis, reconstructive surgeries, ablations, and ischemia.

In one embodiment, a placental product of the present invention is usedin a tissue graft procedure. Optionally, the placental product isapplied to a portion of the graft which is then attached to a biologicalsubstrate (e.g. to promote healing and/or attachment to the substrate).By way of non-limiting example, tissues such as skin, cartilage,ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenterand sinew can be used as tissue graft.

In one embodiment, a placental product is used in a tendon or ligamentsurgery to promote healing of a tendon or ligament. Optionally, theplacental product is applied to portion of a tendon or ligament which isattached to a bone. The surgery can be any tendon or ligament surgery,including, e.g. knee surgery, shoulder, leg surgery, arm surgery, elbowsurgery, finger surgery, hand surgery, wrist surgery, toe surgery, footsurgery, ankle surgery, and the like. For example, the placental productcan be applied to a tendon or ligament in a grafting or reconstructionprocedure to promote fixation of the tendon or ligament to a bone.

Through the insight of the inventors, it has surprisingly beendiscovered that placental products of the present invention providesuperior treatment (e.g. healing time and/or healing strength) fortendon and ligament surgeries. Tendon and ligament surgeries can involvethe fixation of the tendon or ligament to bone. Without being bound bytheory, the present inventors believe that osteogenic and/orchondrogenic potential of MSCs in the present placental productspromotes healing process and healing strength of tendons or ligaments.The present inventors believe that the present placental productsprovide an alternative or adjunctive treatment to periosteum-basedtherapies. For example, useful periosteum based treatments are describedin Chen et al. (“Enveloping the tendon graft with periosteum to enhancetendon-bone healing in a bone tunnel: A biomechanical and histologicstudy in rabbits”; Arthroscopy. 2003 March; 19(3):290-6), Chen et al.(“Enveloping of periosteum on the hamstring tendon graft in anteriorcruciate ligament reconstruction”; Arthroscopy. 2002 May-June;18(5):27E), Chang et al. (“Rotator cuff repair with periosteum forenhancing tendon-bone healing: a biomechanical and histological study inrabbits”; Knee Surgery, Sports Traumatology, Arthroscopy Volume 17,Number 12, 1447-1453), each of which are incorporated by reference.

As non-limiting example of a method of tendon or ligament surgery, atendon is sutured to and/or wrapped or enveloped in a placental membraneand the tendon is attached to a bone. Optionally, the tendon is placedinto a bone tunnel before attached to the bone.

In one embodiment, the tendon or ligament surgery is a graft procedure,wherein the placental product is applied to the graft. Optionally, thegraft is an allograft, xenograft, or an autologous graft.

In one embodiment, the tendon or ligament surgery is repair of a tornligament or tendon, wherein the placental product is applied to the tornligament or tendon.

Non-limiting examples of tendons to which a placental product can beapplied include a digitorum extensor tendon, a hamstring tendon, a biceptendon, an Achilles Tendon, an extensor tendon, and a rotator cufftendon.

In one embodiment, a placental product of the present invention is usedto reduce fibrosis by applying the placental product to a wound site.

In one embodiment, a placental product of the present invention is usedas an anti-adhesion wound barrier, wherein the placental product isapplied to a wound site, for example, to reduce fibrosis (e.g.postoperative fibrosis).

Non-limiting examples of wound sites to which the placental product canbe applied include those that are surgically induced or associated withsurgery involving the spine, laminectomy, knee, shoulder, or childbirth, trauma related wounds or injuries, cardiovascular procedures,angiogenesis stimulation, brain/neurological procedures, burn and woundcare, and ophthalmic procedures. For example, optionally, the wound siteis associated with surgery of the spine and the stromal side of theplacental product is applied to the dura (e.g. the stromal side facingthe dura). Direction for such procedures, including the selection ofwound sites and/or methodologies, can be found, for example, in WO2009/132186 and US 2010/0098743, which are hereby incorporated byreference.

A placental product of the present invention can optionally be used toreduce adhesion or fibrosis of a wound. Postoperative fibrosis is anatural consequence of all surgical wound healing. By example,postoperative peridural adhesion results in tethering, traction, andcompression of the thecal sac and nerve roots, which cause a recurrenceof hyperesthesia that typically manifests a few months after laminectomysurgery. Repeated surgery for removal of scar tissue is associated withpoor outcome and increased risk of injury because of the difficulty ofidentifying neural structures that are surrounded by scar tissue.Therefore, experimental and clinical studies have primarily focused onpreventing the adhesion of scar tissue to the dura matter and nerveroots. Spinal adhesions have been implicated as a major contributingfactor in failure of spine surgery. Fibrotic scar tissue can causecompression and tethering of nerve roots, which can be associated withrecurrent pain and physical impairment.

Without being bound by theory, the present inventors believe thatplacental products taught herein are useful to reduce adhesion orfibrosis of a wound, at least in part, because the placental productscan perform the very critical function in-situ of providing aimmunoprivileged environment (i.e. relatively high resistance againstimmune responses) in the human development process. One advantage of thewound dressings and processes of the present invention is that ananti-adhesion barrier is provided which can be used to prevent adhesionsfollowing surgery, and in particular following back surgery.

In the preceding paragraphs, use of the singular may include the pluralexcept where specifically indicated. As used herein, the words “a,”“an,” and “the” mean “one or more,” unless otherwise specified. Inaddition, where aspects of the present technology are described withreference to lists of alternatives, the technology includes anyindividual member or subgroup of the list of alternatives and anycombinations of one or more thereof.

The disclosures of all patents and publications, including publishedpatent applications, are hereby incorporated by reference in theirentireties to the same extent as if each patent and publication werespecifically and individually incorporated by reference.

It is to be understood that the scope of the present technology is notto be limited to the specific embodiments described above. The presenttechnology may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

Likewise, the following examples are presented in order to more fullyillustrate the present technology. They should in no way be construed,however, as limiting the broad scope of the technology disclosed herein.

The presently described technology and its advantages will be betterunderstood by reference to the following examples. These examples areprovided to describe specific embodiments of the present technology. Byproviding these specific examples, it is not intended limit the scopeand spirit of the present technology. It will be understood by thoseskilled in the art that the full scope of the presently describedtechnology encompasses the subject matter defined by the claimsappending this specification, and any alterations, modifications, orequivalents of those claims.

EXAMPLES

Other features and embodiments of the present technology will becomeapparent from the following examples which are given for illustration ofthe present technology rather than for limiting its intended scope.

Example 1 Characterization of Placental Membranes

Characterization of cells in placental membranes by FluorescenceActivated Cell Sorting (FACS) demonstrated the presence of stromal cells(Mesenchymal Stem Cell-like cells) in addition to fetal epithelial cellsand fibroblasts in amniotic and/or chorionic membranes.

One unique characteristic of the presently disclosed placental productsis the presence of MSCs, which have been shown to be one of three typesof cells (in addition to epithelial cells and fibroblasts) that areimportant for wound healing. Placental membranes secrete a variety offactors involved in wound healing such as angiogenic factors, factorssupporting proliferation and migration of epithelial cells andfibroblasts, factors attracting endothelial stem cells from bloodcirculation to the wound site, antibacterial factors, and others.

Evaluation of proteins secreted by examplary placental products of theinvention in comparison to Apligraf and Dermagraft demonstrated a numberof growth factors present in the tested products that are involved inwound healing. Examples are Vascular Endothelial Growth Factor (VEGF),Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor (TGF)and others. However, several unique factors including Epidermal GrowthFactor (EGF), which is one of the key factors for wound healing, arepresent in placental membranes and absent in Apligraf and Dermagraft.Also, placental membranes have a favorable protease-to-proteaseinhibitor ratio for wound healing. In an in vitro model of wound healing(cell migration assay, disclosed herein), the present inventors havedemonstrated that placental membranes secrete factors promoting cellmigration that will support wound closure.

Example 2 Exemplary Manufacturing Process of a Placental Product

In one embodiment, the present application discloses a procedure formanufacturing chorionic membranes from placenta post partum.

Example 2.1 Exemplary Manufacturing Process of Chorionic MembraneProduct

One method of manufacturing a placental product comprising a chorionicmembrane according to the presently disclosed manufacturing procedure isas follows:

-   a. Remove umbilical cord close to placental surface,-   b. Blunt dissect of the amnion to placental skirt,-   c. Flip placenta over and completely remove amnion,-   d. Remove chorion by cutting around placental skirt,-   e. Rinse the chorionic membrane in PBS to remove red blood cells,-   f. Rinse the chorionic membrane once with 11% ACD-A solution to    assist in red blood cell removal,-   g. Rinse the chorionic membrane PBS to remove ACD-A solution,-   h. Treat chorion in 0.5% dispase solution at 37° C.±2° C. for 30-45    minutes, optionally, during dispase incubation period, use PBS to    remove any remaining blood from the amnion,-   i. When dispase treatment is complete, rinse chorion with PBS to    remove dispase solution,-   j. Gently remove trophoblast layer from the chorion, for example, by    scraping (e.g. with finger),-   k. Place chorion into a bottle containing antibiotic solution and    incubate at 37° C.±2° C. for 24-28 hrs,-   l. Remove bottle from the incubator and rinse each membrane with PBS    to remove antibiotic solution,-   m. Mount chorion on reinforced nitrocellulose paper and cut to size,-   n. Place each piece into an FP-90 cryobag and heat seal,-   o. Add 50 mL cryopreservation solution to the bag through a syringe    and remove any air trapped within the bag with the syringe,-   p. Tube seal the solution line on the FP-90 bag,-   q. Place filled bag into secondary bag and heat seal,-   r. Place unit into packaging carton,-   s. Refrigerate at 2-8° C. for 30-60 minutes, Freeze at −80° C.±5° C.    inside a Styrofoam container.

Example 2.2 Exemplary Manufacturing Process of Product ComprisingChorionic Membrane and Amniotic Membrane

One method of manufacturing a placental product comprising a chorionicmembrane and an amniotic membrane according to the presently disclosedmanufacturing procedure is as follows:

-   a. Remove umbilical cord close to placental surface,-   b. Blunt dissect of the amnion to placental skirt,-   c. Flip placenta over and completely remove amnion,-   d. Remove chorion by cutting around placental skirt,-   e. Rinse both membranes in PBS to remove red blood cells,-   f. Rinse both membranes once with 11% ACD-A solution to assist in    red blood cell removal,-   g. Rinse both membranes with PBS to remove ACD-A solution,-   h. Treat chorion in 0.5% dispase solution at 37° C.±2° C. for 30-45    minutes, optionally, during dispase incubation period, use PBS to    remove any remaining blood from the amnion,-   i. Gently remove the connective tissue layer from the amnion,-   j. Place the amnion in PBS and set aside,-   k. When dispase treatment is complete, rinse chorion with PBS to    remove dispase solution,-   l. Gently remove trophoblast layer from the chorion,-   m. Place the amnion and chorion each into a bottle containing    antibiotic solution and incubate at 37° C.±2° C. for 24-28 hrs,-   n. Remove bottles from the incubator and rinse each membrane with    PBS to remove antibiotic solution,-   o. Mount amnion (epithelial side up) or chorion on reinforced    nitrocellulose paper and cut to size,-   p. Place each piece into an FP-90 cryobag and heat seal,-   q. Add 50 mL cryopreservation solution to the bag through a syringe    and remove any air trapped within the bag with the syringe,-   r. Tube seal the solution line on the FP-90 bag,-   s. Place filled bag into secondary bag and heat seal,-   t. Place unit into packaging carton,-   u. Refrigerate at 2-8° C. for 30-60 minutes, Freeze at −80° C.±5° C.    inside a Styrofoam container.

Example 2.3 Exemplary Placental Product Manufacturing Process

One method manufacturing a placental product comprising a chorionicmembrane according to the presently disclosed manufacturing procedurewas as follows:

The placenta was processed inside a biological safety cabinet. Theumbilical cord was first removed, and the amniotic membrane was peeledfrom the underlying chorionic membrane using blunt dissection.Subsequently, the chorion was removed by cutting around the placentalskirt on the side opposite of the umbilical cord. The chorion on theumbilical side of the placenta was not removed due to thevascularization on this side. The chorionic membrane was rinsed withphosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, N.Y.)to remove gross blood clots and any excess blood cells. The membrane wasthen washed with 11% anticoagulant citrate dextrose solution (USP)formula A (ACD-A) (Baxter Healthcare Corp., Deerfield, Ill.) in saline(Baxter Healthcare Corp., Deerfield, Ill.) to remove remaining bloodcells.

The chorion was then incubated in 200 mL of a 0.5% dispase (BDBiosciences, Bedford, Mass.) solution in Dulbecco's Modified Eaglesmedia (DMEM) (Lonza, Walkersville, Md.) at 37° C.±2° C. for 30-45minutes to digest the connective tissue layer between the chorion andadjacent trophoblast layer. Once the chorion incubation period wascomplete, the chorion was rinsed with PBS to remove the dispasesolution. Subsequently, the trophoblast layer was removed by gentlypeeling or scraping away these maternal decidual cells.

The chorion was then disinfected in 500 mL of antibiotic solutionconsisting of gentamicin sulfate (50 μg/mL) (Abraxis PharmaceuticalProducts, Schaumburg, Ill.), vancomycin HCl (50 μg/mL) (Hospira Inc.,Lake Forest, Ill.), and amphotericin B (2.5 μg/mL) (Sigma Aldrich, St.Louis, Mo.) in DMEM at 37° C.±2° C. for 24-28 hours. Vented caps wereused with the incubation flasks. After the incubation period, themembrane was washed with PBS to remove any residual antibiotic solution.

The membrane was mounted on Optitran BA-S 85 reinforced nitrocellulosepaper (Whatman, Dassel, Germany) and cut to the appropriate size priorto packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem,N.C.). Once the membrane unit was placed into the FP-90 cryobag and thecryobag was heat sealed, 50 mL of a cryopreservation solution containing10% dimethyl sulfoxide (DMSO) (Bioniche Teo. Inverin Co., Galway,Ireland) and 5% human serum albumin (HSA) (Baxter, West Lake Village,Calif.) in PlasmaLyte-A (Baxter Healthcare Corp., Deerfield, Ill.) wereadded through the center tubing line. Any excess air was removed, andthe tubing line was subsequently sealed.

The FP-90 cryobag was placed into a mangar bag (10 in.×6 in.) (MangarIndustries, New Britain, Pa.), which was then heat sealed. The mangarbag was placed into a packaging carton (10.5 in.×6.5 in.×0.6 in.)(Diamond Packaging, Rochester, N.Y.). All cartons were refrigerated at2-8° C. for 30-60 minutes prior to freezing at −80° C.±5° C. inside aStyrofoam container.

Example 2.4 Exemplary Manufacturing Process of a Placental ProductComprising Chorionic Membrane and Amniotic Membrane

One method of manufacturing a placental product comprising a chorionicmembrane product and an amniotic membrane product according to thepresently disclosed manufacturing procedure was as follows:

The placenta was processed inside a biological safety cabinet. Theumbilical cord was first removed, and the amniotic membrane was peeledfrom the underlying chorionic membrane using blunt dissection.Subsequently, the chorion was removed by cutting around the placentalskirt on the side opposite of the umbilical cord. The chorion on theumbilical side of the placenta was not removed due to thevascularization on this side. Both membranes were rinsed with phosphatebuffered saline (PBS) (Gibco Invitrogen, Grand Island, N.Y.) to removegross blood clots and any excess blood cells. The membranes were thenwashed with 11% anticoagulant citrate dextrose solution (USP) formula A(ACD-A) (Baxter Healthcare Corp., Deerfield, Ill.) in saline (BaxterHealthcare Corp., Deerfield, Ill.) to remove remaining blood cells.

The chorion was then incubated in 200 mL of a 0.5% dispase (BDBiosciences, Bedford, Mass.) solution in Dulbecco's modified eaglesmedia (DMEM) (Lonza, Walkersville, Md.) at 37° C.±2° C. for 30-45minutes to digest the connective tissue layer between the chorion andadjacent trophoblast layer. During this incubation period, the stromalside of the amnion was cleaned by gently scraping away any remainingconnective tissue. Once the chorion incubation period was complete, thechorion was rinsed with PBS to remove the dispase solution.Subsequently, the trophoblast layer was removed by gently peeling orscraping away these maternal decidual cells.

The amnion and chorion were then each disinfected in 500 mL ofantibiotic solution consisting of gentamicin sulfate (50 μg/mL) (AbraxisPharmaceutical Products, Schaumburg, Ill.), vancomycin HCl (50 μg/mL)(Hospira Inc., Lake Forest, Ill.), and amphotericin B (2.5 μg/mL) (SigmaAldrich, St. Louis, Mo.) in DMEM at 37° C.±2° C. for 24-28 hours. Ventedcaps were used with the incubation flasks. After the incubation period,the membranes were washed with PBS to remove any residual antibioticsolution.

The membranes were mounted on Optitran BA-S 85 reinforced nitrocellulosepaper (Whatman, Dassel, Germany) and cut to the appropriate size priorto packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem,N.C.). For the amnion, the stromal side was mounted towards thenitrocellulose paper. Once a membrane unit was placed into the FP-90cryobag and the cryobag was heat sealed, 50 mL of a cryopreservationsolution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. InverinCo., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, WestLake Village, Calif.) in PlasmaLyte-A (Baxter Healthcare Corp.,Deerfield, Ill.) were added through the center tubing line. Any excessair was removed, and the tubing line was subsequently sealed.

The FP-90 cryobag was placed into a mangar bag (10 in.×6 in.) (MangarIndustries, New Britain, Pa.), which was then heat sealed. The mangarbag was placed into a packaging carton (10.5 in.×6.5 in.×0.6 in.)(Diamond Packaging, Rochester, N.Y.). All cartons were refrigerated at2-8° C. for 30-60 minutes prior to freezing at −80° C.±5° C. inside aStyrofoam container.

Example 3 Quantitative Evaluation of Cell Number and Cell Viabilityafter Enzymatic Digestion of Placental Membranes

Amnion and chorion membranes and present placental products (from above)were evaluated for cell number and cell viability throughout theprocess. These analyses were performed on fresh placental tissue (priorto the antibiotic treatment step), placental tissue post antibiotictreatment, and product units post thaw. Cells were isolated from theplacental membranes using enzymatic digestion. For the frozen productunits, the FP-90 cryobags were first removed from the packaging cartonsand mangar bags. Then the FP-90 cryobags were thawed for 2-3 minutes ina room temperature water bath. Early experiments involved the use of a37° C.±2° C. water bath. After thaw, the placental membranes wereremoved from the FP-90 cryobag and placed into a reservoir containingsaline (Baxter Healthcare Corp., Deerfield, Ill.) for a minimum of 1minute and a maximum of 60 minutes. Each membrane was detached from thereinforced nitrocellulose paper prior to digestion.

Amniotic membranes were digested with 40 mL of 0.75% collagenase(Worthington Biochemical Corp., Lakewood, N.J.) solution at 37° C.±2° C.for 20-40 minutes on a rocker. After collagenase digestion, the sampleswere centrifuged at 2000 rpm for 10 minutes. The supernatant wasremoved, and 40 mL of 0.05% trypsin-EDTA (Lonza, Walkersville, Md.) wereadded and incubated at 37° C.±2° C. for an additional 5-15 minutes on arocker. The trypsin was warmed to 37° C.±2° C. in a water bath prior touse. After trypsin digestion, the suspension was filtered through a 100μm cell strainer nylon filter to remove any debris. Centrifugation at2000 rpm for 10 minutes was performed, and supernatant was removed. Cellpellets were reconstituted with a volume of PlasmaLyte-A that wasproportional to the pellet size, and 20 μL of the resuspended cellsuspension were mixed with 80 μL of trypan blue (Sigma Aldrich, St.Louis, Mo.) for counting. The cell count sample was placed into ahemocytometer and evaluated using a microscope.

Chorionic membranes were digested with 25 mL of 0.75% collagenasesolution at 37° C.±2° C. for 20-40 minutes on a rocker. Aftercollagenase digestion, the suspension was filtered through a 100 μm cellstrainer nylon filter to remove any debris. Centrifugation at 2000 rpmfor 10 minutes was performed, and supernatant was removed. Cell pelletswere reconstituted with a volume of PlasmaLyte-A that was proportionalto the pellet size, and 20 μL of the resuspended cell suspension weremixed with 80 μL of trypan blue for counting. The cell count sample wasplaced into a hemocytometer and evaluated using a microscope.

Placenta membranes were analyzed prior to any processing to determinethe initial characteristics of the membranes. Table 1 contains theaverage cell count per cm² and cell viability values for the amnioticand chorionic membranes from 32 placenta lots.

The average cell count per cm² for the amniotic membrane was 91,381cells with a corresponding average cell viability of 84.5%. For thechorionic membrane, the average cell count per cm² was 51,614 cells witha corresponding cell viability of 86.0%.

These data illustrate cell numbers that are useful with certainembodiments of the present invention; e.g. a placental productcomprising a chorionic membrane containing about 20,000 to about 200,000cells/cm².

Since the amniotic membrane consists of epithelial cells and stromalcells, experiments were conducted to determine the ratio of epithelialcells to stromal cells. Amniotic membranes from 3 placenta lots wereanalyzed. First, a 5 cm×5 cm piece of amniotic membrane was digestedwith approximately 25 mL of 0.05% Trypsin-EDTA (Lonza, Walkersville,Md.) at 37° C.±2° C. in a water bath for 30 minutes. After theincubation step, epithelial cells were removed by gently scraping thecells from the membrane. After rinsing with PBS (Gibco Invitrogen, GrandIsland, N.Y.), the membrane was subsequently digested in the same manneras chorionic membrane (described above). In addition, another intact 5cm×5 cm piece of amniotic membrane was digested using the standardprocedure (described above) to determine the total number of cells. Thepercentage of stromal cells was then determined by dividing the cellcount from the amniotic membrane with the epithelial cells removed withthe cell count from the intact membrane.

Results indicate that 19% of the total cells were stromal cells.Therefore, approximately 17,362 stromal cells were present in amnioticmembrane with approximately 74,019 epithelial cells. These dataindicated that there are approximately 3 times more stromal cells inchorionic membranes as compared to amniotic membranes. This ratio isconsistent with certain embodiments of the present invention thatprovide a placental product comprising a chorionic membrane and anamniotic membrane, wherein the chorionic membrane comprises about 2 toabout 4 times more stromal cells relative to the amniotic membrane.

TABLE 1 Cell count per cm² and cell viability values from freshplacental tissue from 32 donors. Cell Count Cell Membrane Statistics percm² Viability Amnion Average 91,381 84.5% SD 49,597 3.7% Chorion Average51,614 86.0% SD 25,478 4.7%

The second point in the manufacturing process where cell count and cellviability values were assessed was after the antibiotic treatment step.Table 2 provides the results from these analyses. Cell recoveries fromthis step for the amniotic membrane and the chorionic membrane were87.7% and 70.3%, respectively.

TABLE 2 Cell count per cm², cell viability, and process (antibiotictreatment) cell recovery values for post antibiotic placental tissuefrom 28 donors. Cell Count Cell Process Cell Membrane Statistics per cm²Viability Recovery Amnion Average 75,230 84.4% 87.7% SD 46,890 4.2%49.4% Chorion Average 33,028 85.6% 70.3% SD 18,595 4.4% 31.1%

Example 4 Development of a Placental Product Cryopreservation Procedure

Cryopreservation is a method that provides a source of tissues andliving cells. A main objective of cryopreservation is to minimize damageto biological materials during low temperature freezing and storage.Although general cryopreservation rules are applicable to all cells,tissues, and organs, optimization of the cryopreservation procedure isrequired for each type of biological material. The present applicationdiscloses a cryopreservation procedure for placental membrane productsthat can selectively deplete immunogenic cells from the placentalmembranes; and preserve viability of other beneficial cells that are theprimary source of factors for the promotion of healing.

During cryopreservation method development for placental membranes, thepresent inventors evaluated key parameters of cryopreservation includingvolume of cryopreservative solution, effect of tissue equilibrationprior to freezing, and cooling rates for a freezing procedures.

Acceptance of tissue allografts in the absence of immunosuppression willdepend on the number of satellite immune cells present in the tissue.Cryopreservation is an approach which can be utilized to reduce tissueimmunogenicity. This approach is based on differential susceptibility ofdifferent cell types to freezing injury in the presence of DMSO;leukocytes are sensitive to fast cooling rates. The freezing rate of 1°C./min is considered optimal for cells and tissues including immunecells. Rapid freezing rates such as 60-100° C./min eliminate immunecells. However, this type of procedure is harmful to other tissue cells,which are desirable for preservation according to the present invention.The developed cryopreservation procedure utilized a cryopreservationmedium containing 10% DMSO, which is a key component protecting cellsfrom destruction when water forms crystals at low temperatures. Thesecond step of cryopreservation was full equilibration of placentalmembrane in the cryopreservation medium, which was achieved by soakingmembranes in the cryopreservation medium for 30-60 min at 4° C. Thisstep allowed DMSO to penetrate the placental tissues. Although there aredata in the literature showing that tissue equilibration prior tofreezing affects survival of immune cells (Taylor & Bank, Cryobiology,1988, 25:1), it was an unexpected finding that 30-60 min placentalmembrane equilibration in a DMSO-containing solution at 2-8° C.selectively increases sensitivity of immune cells to freezing (incomparison to therapeutic cells) so that these type of cells areselectively depleted during the freezing process (e.g. 1° C./minfreezing rate).

For example, CD14+ macrophages are selectively killed relative totherapeutic cells such as hMSCs and/or fibroblasts.

Temperature mapping experiments were performed to analyze thetemperature profiles of potential cryopreservation conditions for themembrane products. These results are illustrated in FIG. 1. Eight (8)FP-90 cryobags were filled with either 20 mL or 50 mL ofcryopreservation solution, and temperature probes were placed insideeach cryobag. The first set of parameters (conditions 1 through 4 ofFIG. 1a through FIG. 1d , respectively) involved a 30-minuterefrigeration (2-8° C.) step prior to freezing (−80° C.±5° C.). Inaddition, the analysis involved freezing of the cryobags either inside aStyrofoam container or on the freezer shelf. The second set ofparameters (conditions 5 through 8 of FIG. 1e through FIG. 1h ,respectively) involved direct freezing (−80° C.±5° C.) of the cryobagseither inside a Styrofoam container or on the freezer shelf. The resultsindicated that condition 6 and condition 2 exhibited the most gradualtemperature decreases. Gradual temperature decreases are typicallydesired in order to preserve cell viability. The difference betweencondition 6 and condition 2 was that condition 2 included a 30-minuterefrigeration step. Therefore, the decrease in temperature from thestart of freezing to −4° C., where latent heat evolution upon freezingoccurs, was examined further. For condition 6, the rate of cooling wasapproximately −1° C./minute during this period. The rate of cooling forcondition 2 was approximately −0.4° C./minute during the same timeframe.Therefore, condition 2 was selected for incorporation into anon-limiting cryopreservation process since slower rates of cooling aregenerally desired to maintain optimal cell viability.

FIG. 2 depicts the effects of cryopreservation solution volume onprocess (cryopreservation) cell recovery for the chorionic membrane. Theanalysis of the 10 mL cryopreservation solution volume involved 5placenta lots, and the analysis of the mL cryopreservation solutionvolume included 3 lots. For the 50 mL cryopreservation solution volume,16 placenta lots were analyzed.

As depicted in FIG. 2, the 50 mL volume of cryopreservation solutionvolume provided superior cell recovery compared to that of the 10 ml and20 ml. These data indicate that a cryopreservation medium volume ofgreater than 20 mL such as about 50 mL or more can provide superiorplacental product according to the present invention.

Experiments were conducted to evaluate different potential freezingconditions to maximize cell recovery after the cryopreservation process.

FIG. 3 includes these results, depicting the effects of refrigerationtime and freezing parameters on process (cryopreservation) cell recoveryfor the chorionic membrane. Three conditions were analyzed. Theseconditions were also linked to the temperature mapping studies. Thefirst condition involved directly freezing the product unit on a shelfwithin the freezer (−80° C.±5° C.). The second condition also containeda direct freeze, but the product unit was placed into a Styrofoamcontainer within the freezer. The third condition included arefrigeration (2-8° C.) period of 30 minutes prior to the freezing step.For the amniotic membrane, 3 placenta lots were evaluated. Two (2)placenta lots were analyzed for the chorionic membrane. Resultsindicated that the third condition was optimal for both membrane types.As depicted in

FIG. 3, a refrigeration period at least about 30 min provided the bestcell recovery.

Cryopreservation parameters are assessed for the amniotic and chorionicmembranes and summarized in Table 3 and Table 4. The evaluation of thecell recoveries and cell viabilities from these experiments resulted inthe selection of the final parameters for the manufacturing process. Inaddition, all average cell viability values were ≥70%.

TABLE 3 Post thaw cell count per cm², cell viability, and process(cryopreservation) cell recovery values for the chorionic membrane. CellProcess Con- Count Cell Cell dition Sta- per Vi- Re- Parameter Testedtistics cm² ability covery Refrigerate All Average 23,217 87.3% 102.8% at 2-8° C. con- SD 9,155  4.1% 65.5% for 30-60 ditions N 27 27 27 minand freeze at −80° C. ± 5° C. Dispase 30 min Average 22,354 85.7% 81.1%No decrease treatment SD 9,505  5.1% 32.4% in process N 24 24 24 cell 45min Average 27,125 90.6% 172.6%  recovery SD 7,963  2.2% 101.2%  for the45 N 6 6 6 min treat- ment. A 30-45 min range was established. Re- 30min Average 23,815 86.8% 102.2%  The process frigeration SD 9,681  5.2%68.8% recovery time N 25 25 25 value was interval 60 min Average 20,77385.8% 84.9% >80% for SD 7,356  4.7% 14.4% the 60 min N 5 5 5 timeinterval. A 30-60 min range was established. Thawing 37° C. ± Average33,360 85.9% 114.7%  No tem- 2° C. SD 8,497  4.0% 38.1% significantperature water N 5 5 5 difference bath found in Room Average 21,29886.8% 96.3% process cell temp SD 8,189  5.3% 67.2% recovery. water N 2525 25 The room bath temp condition was selected for logistical reasons.Holding 1-15 Average 23,733 86.6% 100.6%  No period min SD 9,674  5.1%67.0% significant after N 26 26 26 difference transfer 1 hr Average20,550 87.0% 91.4% found in into saline SD 6,575  4.8% 32.0% processcell N 4 4 4 recovery. Membranes can be held in saline for up to 1 hr.Tissue 5 cm × Average 23,391 86.1% 99.6% No decrease size 5 cm SD 8,865 5.0% 58.7% in process N 23 23 23 cell 2 cm × Average 23,036 88.4% 98.7%recovery 2 cm SD 11,362  5.0% 81.3% from the N 7 7 7 5 cm × 5 cm productto the 2 cm × 2 cm product. Both sizes were acceptable for use. Notes:cm = centimeter; min = minutes; temp = temperature; hr = hour, SD =standard deviation; N = number

TABLE 4 Post thaw cell count per cm², cell viability, and process(cryopreservation) cell recovery values for the amniotic membrane CellProcess Con- Count Cell Cell dition Sta- per Vi- Re- Comments/ ParameterTested tistics cm² ability covery Conclusions Refrigerate All Average55,709 83.4% 64.2% Overall at 2-8° C. con- SD 45,210  4.4% 22.5%assessment for 30-60 ditions N 32 32 32 min and freeze at −80° C. ± 10°C. Re- 30 min Average 52,173 83.1% 63.7% No frigeration SD 39,750  4.5%21.4% significant time N 26 26 26 difference interval 60 min Average71,033 85.0% 66.5% found in SD 66,525  3.9% 29.3% process cell N 6 6 6recovery. A 30-60 min range was established. Thawing 37° C. ± Average48,524 83.3% 64.0% No tem- 2° C. SD 27,804  1.7% 34.4% significantperature water N 7 7 7 difference bath Average 57,721 83.5% 64.3% foundin Room SD 49,271  4.9% 19.0% process cell temp N 25 25 25 recovery.water The room bath temp condition was selected for logistical reasons.Holding 1-15 Average 50,873 83.1% 65.0% No period after min SD 38,969 3.9% 24.2% significant transfer N 26 26 26 difference into 1 hr Average76,667 85.1% 61.0% found in saline SD 66,565  6.2% 14.3% process cell N6 6 6 recovery. Membranes can be held in saline for up to 1 hr. Tissuesize 5 cm × Average 58,431 83.3% 62.8% No decrease 5 cm SD 47,603  4.5%21.7% in process N 28 28 28 cell 2 cm × Average 36,656 84.4% 73.9%recovery 2 cm SD 13,175  3.4% 29.5% from the N 4 4 4 5 cm × 5 cm productto the 2 cm × 2 cm product. Both sizes were acceptable for use.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising a chorionicmembrane containing about 20,000 to about 60,000 or to about 200,000cells/cm².

Example 5 Qualitative Evaluation of Cell Viability by Tissue Staining

The amniotic and chorionic membranes were stained using a LIVE/DEAD®Viability/Cytotoxicity kit (Molecular Probes Inc., Eugene, Oreg.) toqualitatively assess cell viability. Staining was performed as per themanufacturer's protocol. Membrane segments of approximately 0.5 cm×0.5cm were used. Evaluation of stained membranes was performed using afluorescent microscope. An intense uniform green fluorescence indicatedthe presence of live cells, and a bright red fluorescence indicated thepresence of dead cells. Images of fresh amniotic and chorionic membranesas well as cryopreserved amniotic and chorionic membranes demonstratedthat the manufacturing process did not alter the phenotypiccharacteristics of the membranes and the proportion of viable cell types(epithelial and stromal cells) in the membranes post thaw.

FIG. 4 shows representative images of the live/dead staining of theepithelial layer of fresh amniotic membrane (A); epithelial layer ofcryopreserved amniotic membrane (B); stromal layer of fresh amnioticmembrane (C); stromal layer of cryopreserved amniotic membrane (D);fresh chorionic membrane (E); and cryopreserved chorionic membrane (F).Live cells are green, and dead cells are red.

Example 6 Placental Tissue Immunogenicity Testing

One unique feature of the human chorion is the absence of fetal bloodvessels that prevent mobilization of leukocytes from fetal circulation.On the fetal side, macrophages resident in the chorioamniotic mesodermallayer represent the only population of immune cells. Thus, fetalmacrophages present in the chorion are a major source of tissueimmunogenicity, as such the chorion is considered immunogenic. In astudy where the amnion was used together with the chorion for plasticrepair of conjunctival defects, the success rate was low (De Roth ArchOphthalmol, 1940, 23: 522). Without being bound by theory, the presentinventors believe that removal of CD14+ cells from placental membraneseliminates activation of lymphocytes in vitro. In addition to thepresence of fetal macrophages, the present inventors believe thatimmunogenicity of chorion can be mediated by contamination of bloodcells coming from the maternal trophoblast, which contains bloodvessels. Thus, the processing of placental membrane for clinical use canbe enhanced by purification of the chorion from maternal trophoblastsand selective elimination of all CD14+ fetal macrophages. Immunogenicitytesting can be used to characterize a chorion-derived product as safeclinical therapeutics. For example, two bioassays can be used to testimmunogenicity of manufactured placental products: Mixed LymphocyteReaction (MLR) and Lipopolysaccharide (LPS)-induced Tumor NecrosisFactor (TNF)-α secretion.

Example 7 Mixed Lymphocyte Reaction (MLR)

An MLR is a widely used in vitro assay to test cell and tissueimmunogenicity. The assay is based on the ability of immune cells(responders) derived from one individual to recognize allogeneic HumanLeukocyte Antigen (HLA) and other antigenic molecules expressed on thesurface of allogeneic cells and tissues (stimulators) derived fromanother individual when mixed together in a well of an experimentaltissue culture plate. The response of immune cells to stimulation byallogeneic cells and tissues can be measured using a variety of methodssuch as secretion of particular cytokines (e.g., Interleukin (IL-2),expression of certain receptors (e.g., IL-2R), or cell proliferation,all of which are characteristics of activated immune cells.

Placental tissue samples representing different steps of the presentlydisclosed manufacturing process were used for immunogenicity testing.These samples included amnion with chorion and trophoblast as a startingmaterial and separated choriotrophoblast, chorion, trophoblast, andamnion. Both freshly purified and cryopreserved (final products) tissueswere tested.

For the MLR assay, cells from placental tissues were isolated using 280U/mL of collagenase type II (Worthington, Cat No. 4202). Tissues weretreated with enzyme for 60-90 min at 37° C.±2° C., and the resultingcell suspension was filtered through a 100 μm filter to remove tissuedebris. Single cell suspensions were then centrifuged using a Beckman,TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant wasdiscarded after each wash, and cells were resuspended in 2 mL of DMEM(Invitrogen, Cat No. 11885) and evaluated for cell number and cellviability by counting cells in the presence of Trypan blue dye(Invitrogen, Cat No. 15250-061). For the MLR, placental-derived cellswere mixed with allogeneic hPBMCs at a 1:5 ratio in 24-well cultureplates in DMEM supplemented with 5% fetal bovine serum (FBS) andincubated for 4 days in the incubator containing 5% CO₂, 95% humidity at37° C.±2° C. Human Peripheral Blood Mononuclear Cells (hPBMCs) alonewere used as a negative control, and a mixture of two sets of hPBMCsderived from two different donors was used as a positive MLR control.After 4 days of incubation, cells were collected from wells, lysed usinga lysis buffer (Sigma, Cat No. C2978) supplemented with proteaseinhibitor cocktail (Roche, Cat No. 11836153001), and IL-2R was measuredin cell lysates using the sIL-2R ELISA kit (R&D Systems, Cat No. SR2A00)generally following the manufacturer's protocol. The level of IL-2R is ameasure of activation of T-cells in response to immunogenic moleculesexpressed by allogeneic cells. Results of 2 out of 12 representativeexperiments are shown in FIG. 5 and FIG. 6. Results presented in thesefigures demonstrated that the present application discloses a processfor manufacturing of placental membranes that result in lowimmunogenicity of the final chorionic membrane products.

As depicted in FIG. 5, the manufacturing process serially reducesimmunogenicity of the placental product. Samples representing differentsteps of the manufacturing process Chorion+Trophoblast (CT), Trophoblast(T), Amnion (AM), and Chorion (CM) were co-cultured with hPBMCs for 4days. IL-2sR was measured in cell lysates as a marker of T-cellactivation. Negative control shows a basal level of immune cellactivation: PBMCs derived from one donor were cultured alone. Positivecontrol: a mixture of PBMCs derived from 2 different donors.

As depicted in FIG. 6, selective depletion of immunogenicity resultsfrom the present cryopreservation process of producing the presentplacental products, as evidenced by the significant decrease inimmunogenicity upon cryopreservation.

Example 8 LPS-Induced TNF-α Secretion by Placental Membrane Cells

As described herein, fetal macrophages present in the amnion and chorionare a major source of tissue immunogenicity. Without being bound bytheory, the present inventors believe that removal of CD14+ cells fromplacental membrane eliminates activation of lymphocytes and thatdepletion of allogeneic donor tissue macrophages decreases the level ofinflammatory cytokine secretion and tissue immunogenicity. The inventorsalso believe that reduction of tissue immunogenicity can also be reachedby depletion of TNF-α with anti-TNF-α antibodies or suppression of TNF-αsecretion by IL-10. Macrophages in fetal placental membranes respond tobacteria by secretion of inflammatory cytokines. The secretion of TNF-αby fresh placental membranes in vitro in response to bacterial LPS issignificantly higher in the chorionic membrane. Thus, the presentinventors believe that immunogenicity of placental membranes is mediatedby macrophages, the amount and/or activity of which is higher in thechorionic membrane.

According to the present invention, selective depletion of macrophagesis an optional approach to selectively deplete immunogenicity of theamniotic and chorionic membranes, allowing the use of both allogeneicmembranes for clinical applications. The assay of functional macrophagesin a placental product is used here as an assay for immunogenicitytesting (e.g. in production or prior to clinical use) based on the factsthat: macrophages are the source of immunogenicity in chorionicmembranes. Macrophages in placenta-derived membranes respond tobacterial LPS by secretion of high levels of TNF-α; and TNF-α is acritical cytokine involved in immune response and allograft tissuerejection. Therefore, secretion of TNF-α by placenta-derived membranesin response to LPS is used here to characterize tissue immunogenicityand for pre-use screening.

Example 9 Establishment of Allowed LPS-Induced TNF-α Secretion Level byChorionic Membranes

Data from published reports regarding the level of TNF-α, which isassociated with the absence or an insignificant immune response in avariety of experimental systems, are presented in Table 5. These dataindicate that a TNF-α level below 100 pg/mL correlates with a low immuneresponse. The ability of amniotic and chorionic membranes to produceTNF-α spontaneously and in response to bacteria or bacterial LPS invitro has been shown by a number of investigators. Table 6 summarizessuch data. The lowest spontaneous TNF-α secretion by amniotic membraneof about 70 pg/cm² of the membrane was reported by Fortunato et al. (AmJ Reprod Immunol, 1994, 32:184). All reports also showed that freshplacental membranes secrete large amounts of TNF-α in response tobacteria or bacterial LPS (Table 6), which is attributed to the presenceof viable functional macrophages.

TABLE 5 TNF-α levels associated with the Description ofabsence/reduction experimental system of immune response CommentsReferences IL-10-induced inhibition Mean 260 pg/mL Wang et al., of MLRin vitro. Transplantation, TNF was measured in 2002, 74: 772 tissueculture supernatant by ELISA. MLR using skin tissue Mean 100 pg/mLexplants (0.02 cm² per well) as stimulators in the presence or absenceof IL-10 (skin explant assay). Skin tissue destruction was assessedmicroscopically, and severity was assigned based on histopathologicaltissue damage. Endogeneous TNF ~0.04 U/mL for the TNF activity Shalabyet al., J production in MLR in the negative control and per mg is notImmunol, 1988, presence or absence of MLR in the presence provided. 141:499 anti-TNF antibodies. TNF of anti-TNF levels were assessedantibodies, which using the WEHI-164 correlated with no or cytotoxicityassay. significant inhibition of lymphocyte proliferation TNF levels inBAL fluid of Isograft: below Unmodified Sekine et al., J lung isografts,unmodified detection; AM-depleted allograft: ~45 Immunol, 1997,allograft, and alveolar allograft: ~15 pg/mL pg/mL 159: 4084 macrophages(AM) of BAL (total 75 pg/5 (immunogenic) depleted allograft in rats. mlof BAL) TNF levels in MLR after ~<200 pg/mL TNF Ohashi et al, 48 hoursin the presence correlated with a Clin Immunol, or absence of advancedcomplete inhibition of 2010, 134: 345 glycation end products MLR (MLRinhibitors). TNF levels in MLR. <100 pg/mL TNF in Toungouz et al., MLRwith HLA- Hum Immunol, matched donors 1993, 38: 221 (control, nostimulation) TNF activity in MLR when Negative control ~20 U Unit ofactivity Lomas et al., pieces of cryopreserved of TNF activity; wascalculated Cell Tissue skin allografts (~0.2 cm²) MLR with skin as TNFin Bank, 2004, were incubated with explants: 0-40 U; ng/mL divided 5:23. hPBMCs for 24 hours. Positive control: by OD at 570 Positivecontrol: 600 U nm for the hPBMC + LPS; negative: same hPBMC alone.experimental well Cytokine time course in Optimal TNF after Jordan &Ritter, MLR, including TNF. 24 hours: ~150 pg/mL J Immunol Meth, 2002,260: 1 MLR using skin tissue For no skin Recalculation Dickinson et al.,explants (0.02 cm² per destruction: 0.5-1.1 per 1 cm² of Cytokine, 1994,well) as stimulators in the pg/mL for HLA skin tissue: 6: 141 presenceor absence of compatible lowest TNF anti-TNF antibodies (skinresponders, and 2.6- non- explant assay). Skin 1376 pg/mL forimmunogenic tissue destruction was unmatched MLR level is 100 assessedpg/cm² microscopically, and severity was assigned based onhistopathological tissue damage.

TABLE 6 TNF levels Comments/ secreted by fresh recalculations placentalof the lowest Description of membranes in TNF levels per experimentalsystem culture cm² References TNF secretion by Chorion: basal Lowest TNFZaga et al., Biol “fresh” amnion and 3.3 ± 0.46 ng/cm², level for amnionReprod, 2004, chorion tissues (1.44 LPS-induced: 150- is 1200 pg/cm² 71:1296 cm²) incubated for 24 250 ng/cm² hours in the presence Amnion:basal or absence of LPS 2.5 ± 1.3 ng/cm², (500 ng/mL). LPS-induced: ~50ng/cm² TNF secretion by Basal ~1-2.5 pg/μg Lowest TNF Zaga-Clavellina“fresh” amnion and total protein in the level for amnion et al., Reprodchorion tissues (1.8 medium for both is 800 pg/cm² Biol Endocrinol, cmdiameter disks: 2.5 amnion and chorion; 2007, 5: 46 cm²) incubated for24 E. Coli-induced: hours in the presence amnion → 29.2 or absence of E.Coli (14.5-35.3) pg and in 1 mL medium. chorion → 53.15 (40-94.2) pg perμg total protein TNF secretion by Basal: ~2-64 U/mL 1 unit = ~100-200Paradowska et “fresh” amnion and or 8-10 mg chori- pg/mL; al., Placenta,chorion tissues on; <1 U/mL for 5-7 Lowest TNF 1997, 18: 441 (chorion8-10 mg mg amnion; level for amnion tissue/mL; amnion 5-7LPS-induced: >100 is <100 pg/mL mg/mL, 0.02-0.04 cm²) U/10 mg forchorion corresponding incubated for 20 hours and ~15-17 U/10 to <2500pg/cm² in the presence or mg for amnion absence of LPS (5 μg/mL). TNFsecretion by Amnion: Basal → Lowest TNF Fortunato et al., “fresh” amnion(0.57 40 pg/mL, level for fresh Am J Obstet cm²) in 0.8 mL LPS-induced →amnion is ~70 Gynecol, 1996, incubated for 24 hours 410 pg/mL pg/cm²174: 1855 in the presence or absence of LPS (50 ng/mL). TNF secretion byBasal: Amnion ~7- Amnion is 5-7 mg Thiex et al., “fresh” amnion and 13ng/mL/g tissue); corresponds ~0.02-0.04 Reprod Biol chorion tissues (4Chorion ~18 cm²; Endocrinol, cm²) incubated for 24 ng/mL/g tissue 1 g is~6 cm²; 2009, 7: 117 hours in the presence LPS-induced (1000 Lowest TNFor absence of LPS (1- ng/mL): Amnion ~14 level for amnion 1000 ng/mL)ng/mL/g), is ~1000 pg/cm² Chorion ~27 ng/mL/g

Example 10 LPS-Induced TNF-α Secretion Immunogenicity Assay

2 cm×2 cm pieces of placental derived membranes representing productionintermediates and final placental products were placed in tissue culturemedium and exposed to bacterial LPS (1 μg/mL) for 20-24 hr. After 24hours, tissue culture supernatant were collected and tested for thepresence of TNF-α using a TNF-α ELISA kit (R&D Systems) according to themanufacturer's protocol. Human hPBMCs (SeraCare) known to containmonocytes responding to LPS by secretion of high levels of TNF-α wereused as a positive control in the assay. hPBMCs and placental tissueswithout LPS were also included as controls in the analysis. In thisassay, TNF detected in the culture medium from greater than 70 pg/cm²(corresponding to 280 pg/mL) for both spontaneous and LPS-induced TNF-αsecretion was considered immunogenic.

The low levels of TNF-α and the absence of the response to LPS by AM andCM indicates the absence of viable functional macrophages that are themajor source of immunogenicity for amniotic and chorionic membranes.Results of this assay showed a correlation with the MLR data: tissuesthat produce high levels of TNF-α in response to LPS are immunogenic inthe MLR assay (FIG. 7A and FIG. 7B for TNF-α secretion; FIG. 9, C-MLR).

As depicted in FIG. 7A and FIG. 7B, the manufacturing process seriallyreduces immunogenicity of the placental product. Samples representingdifferent steps of the manufacturing process (Amnion+Chorion+Trophoblast(ACT), Chorion+Trophoblast (CT), Amnion (AM), and Chorion (CM)) wereincubated in the presence of LPS for 24 hr, and after that tissueculture supernatants were tested for the TNF-α by ELISA. Tissuescultured in medium without LPS show the basal level of TNF a secretion.PBMCs, which are known to secrete high levels of TNF, were used as apositive control.

Choriotrophoblast (CT), which secreted high levels of TNF-α (FIG. 7 B),was tested in MLR against two different PBMC donors. CT cells wereco-cultured with PBMCs for 4 days. IL-2αR was measured in cell lysatesas a marker of T-cell activation. Positive control: a mixture of PBMCsderived from 2 different donors.

FIG. 7C shows that preparations producing high levels of TNF-α areimmunogenic. Choriotrophoblast (CT), which secreted high levels of TNF-α(FIG. 7, B), was tested in MLR against two different PBMC donors. CTcells were co-cultured with PBMCs for 4 days. IL-2αR was measured incell lysates as a marker of T-cell activation. Positive control: amixture of PBMCs derived from 2 different donors.

Example 11 Analysis of Placental Cells by FACS

Knowing the cellular composition of chorionic membranes is important fordeveloping a thorough understanding of potential functional roles inwound healing and immunogenicity. Previous reports demonstrated that thechorion contains multiple cell types. In addition to fibroblasts,stromal cells were identified in the chorion. Although there are nofetal blood vessels within the chorionic membranes, it comprisesresident fetal macrophages. The close proximity to maternal bloodcirculation and decidua provide a potential source of immunogenic cells(maternal leukocytes and trophoblast cells) and therefore are apotential source of immunogenicity. To investigate the cellularcomposition of the chorion, FACS analysis was performed.

Example 11.1 FACS Procedure: Single Cell Suspension Preparation

Purified chorionic membranes were used for cellular phenotypic analysisvia FACS. Cells from chorion were isolated using 280 U/mL collagenasetype II (Worthington, Cat No. 4202). Tissues were treated with enzymefor 60-90 min at 37° C.±2° C., and the resulting cell suspension wasfiltered through a 100 μm filter to remove tissue debris. Single cellsuspensions were then centrifuged using a Beckman TJ-6 at 2000 rpm for10 min and washed twice with DPBS. Supernatant was discarded after eachwash, and cells were resuspended in 2 mL of FACS staining buffer(DPBS+0.09% NaN₃+1% FBS).

Example 11.2 Immunolabeling Cells for Specific Cellular Markers

Once the single cell suspension was prepared according to Example 10.1,a minimum of 1×10⁵ cells in 100 μL of FACS staining buffer was treatedwith antibodies labeled with fluorescent dye. Table 7 providesdescriptions of the antibodies and the amounts used. For cell surfacemarkers, cells were incubated for 30 min at room temperature in the darkwith antibodies followed by washing twice with FACS staining buffer bycentrifugation at 1300 rpm for 5 min using a Beckman TJ-6 centrifuge.Cells were then resuspended in 400 μl_of FACS staining buffer andanalyzed using a BD FACSCalibur flow cytometer. To assess cellviability, 10 μL of 7-AAD regent (BD, Cat No. 559925) was added justafter the initial FACS analysis and analyzed again. For intracellularstaining, cells were permeabilized and labeled following themanufacturer's recommendations (BD Cytofix/Cytoperm, Cat No. 554714) andanalyzed using a BD FACSCalibur flow cytometer.

TABLE 7 Description of reagents used for placental cell characterizationby FACS. Volume of Cell marker antibody antibody and solution Cellmarker Cell marker label type Cat No. used type specificity IgG1isotype- BD 559320 5 μL Cell surface Isotype PE control CD105-PE Caltag20 μL Cell surface MSC marker MHCD10504 CD166-PE BD 559263 80 μL Cellsurface MSC marker CD45-PE BD 555483 10 μL Cell surface Hema- topoieticcell marker IgG2a isotype- BD 555574 2 μL Cell surface Isotype PEcontrol CD14-PE BD 555398 20 μL Cell surface Monocyte marker HLA-DR-PEBD 556644 20 μL Cell surface HLA class II specific for antigen-presenting cells IgG1 isotype- BD555748 5 μL Cell surface Isotype FITCcontrol CD86-FITC BD 557343 20 μL Cell surface Immune co- stimulatorymarker CD40-FITC BD 556624 20 μL Cell surface Immune co- stimulatorymarker IgG1 isotype- Dako X0931 10 μL Intracellular Isotype unlabeledcontrol Cytokeratin 7- Dako M7018 2 μL Intracellular Trophoblastunlabeled marker Rabbit anti- Dako F0261 5 μL Intracellular Secondarymouse FITC antibody

Example 12 Phenotypic Analysis of Placental Cells

FACS analysis of single cell suspensions of chorionic membranesdemonstrates that both membranes contain cells expressing markersspecific for mesenchymal stem cells (refer to Table 8), implicating thepresence of stromal cells. In addition, several immunogenic markers,which are more likely expressed on CD14+ placental macrophages, weredetected. The % ranges for different markers are wide. It can beexplained by: 1) high variability in cell number between placentadonors; and 2) technical issues, which include the presence of the highand variable cellular and tissue debris in the cellular suspension.Although debris can be gated out, debris particles that are comparablewith cells by size will affect the accuracy of the calculated % for eachtested marker. In addition, Table 9 provides a FACS analysis of cellsfrom the chorionic membranes that were cultured in 10% FBS in DMEM at37° C.±2° C. until confluency (passage 0 cells). These data demonstratedthat cells derived from chorionic membranes retained a phenotype similarto MSCs after culturing. In conclusion, the presence of stromal cells inplacental tissues was confirmed by FACS analysis.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising a chorionicmembrane containing MSCs.

TABLE 8 Characterization of the cellular composition of placentalmembranes based on selective CD markers. Chorion Marker (% range) MSCMarkers CD105 6.4-78.5 CD166 4.8-51.5 Hematopoietic Cell CD14 0.9-6.1 Markers CD45 4.6-14.7 Immune co-stimulatory HLA-DR  0-14.7 markers CD864.9-22.5 CD40  2-5.8 Trophoblast marker Cytokeratin-7 2.71-23.07

TABLE 9 FACS analysis of cultured cells (passage 0) from placenta lotD16. Cell Surface Marker Chorion (%) CD45 0.53 CD166 82.62 CD105 86.73CD49a 92.26 CD73 94.57 CD41a −0.05 CD34 −0.25 HLA-DR −0.19 CD19 −0.22CD14 −0.27 CD90 98.00

Example 13 Differentiation Capacity of Cells Derived from the ChorionicMembrane

Therapeutic cells, in optional embodiments of the present invention, areadherent, express specific cellular markers such as CD105 and lackexpression of other markers such as CD45, and demonstrate the ability todifferentiate into adipocytes, osteoblasts, and chondroblasts.

The expression of specific cellular markers has already been describedin Example 12. To determine if the cells within the placental productderived from the chorionic membrane can adhere to plastic anddifferentiate into one of the lineages, cells were isolated from theplacental product derived from the chorion as described in thisinvention and cultured at 37° C.±2° C. and expanded.

FIG. 8-A shows a representative image of passage 2 cells, demonstratingthe ability of the cells to adhere to tissue culture plastic. As acomparison, a representative image of MSCs isolated and expanded fromhuman bone marrow aspirate is shown in FIG. 8-B.

Osteogenic differentiation capacity was demonstrated by staining thecultured cells with alkaline phosphatase labeling following themanufacturer's recommendations (BCIP/NBT Alkaline Phosphatase SubstrateKit IV, Vector Laboratories Cat. No. SK-5400). Alkaline phosphatase isan enzyme involved in bone mineralization (Allori et al., TissueEngineering: Part B, 2008, 8:275), and its expression within cells isindicative of osteo-precursor cells (Majors et al., J Orthopaedic Res,1997, 15:546). Staining for alkaline phosphatase is carried out throughan enzymatic reaction with Bromo-4-Chloro-3′-Indolylphosphatep-Toluidine Salt (BCIP) and Nitro-Blue Tetrazolium Chloride (NTP). BCIPis hydrolyzed by alkaline phosphatase to form an intermediate thatundergoes dimerization to produce an indigo dye. The NBT is reduced tothe NBT-formazan by the two reducing equivalents generated by thedimerization. Together these reactions produce an intense, insolubleblack-purple precipitate when reacted with alkaline phosphatase. FIG.8-C shows a representative image of passage 2 cells staining positivelyfor alkaline phosphatase.

Example 14 Live CD45+ FACS Analysis

As CD45 is a general marker for hematopoietic cells and therefore amarker for the presence immunogenic cells, the presence of CD45+ cellsmay correlate well with how immunogenic a tissue may be. An initialstudy indeed showed a correlation between amount of immunogenicity asmeasured via an in vitro MLR assay of placental tissue at various stageswithin the manufacturing process (as described previously), and theamount of CD45+ cells was determined via FACS analysis. As FIG. 9demonstrates, membranes that trigger the expression of high levels ofIL-2sR on hPBMC responders in MLR also contained a high percentage ofCD45+ cells, indicating that immunogenicity of placental membranes canbe correlated with the number of CD45+ cells. Further studies revealed,however, that quantifying CD45+ cells via FACS alone showed highvariability that did not allow for the establishment of a safetythreshold for CD45+ cells in placental membranes. Accordingly, theinventors evaluated whether or not viability of CD45+ cells iscorrelated with immunogenicity.

To eliminate some of the variability in CD45+ measurements via FACS,viability of CD45+ cells was assessed, as dead CD45+ cells do notcontribute to immunogenicity. To ensure an accurate assessment of liveCD45+ cells, a pilot experiment was conducted in which a single cellsuspension of amnion membrane was spiked in with a known concentrationof live CD45+ cells (hPBMCs) ranging from a theoretical 1.25% to 20%(0.75-12%—actual % of the spiked cells) of the total cell concentrationin suspension. Cells were stained with CD45-PE antibody at determinedconcentrations (refer to Table 10), incubated with 7-AAD cell viabilitytest reagent, and analyzed using a BD FACSCalibur. Table 10 demonstratesthat recovery of known amounts of CD45+ cells was not correct (4thcolumn in the table). For example, although 12% of PBMCs was spiked intoa single-cell suspension of amnion membrane, only 4.26% of CD45+ cellswere recovered according to FACS analysis (>60% difference from theactual spike). To correlate with immunogenicity, MLR was also performedin parallel. Briefly, single cell suspensions of amniotic membranespiked with various amounts of live hPBMCs were co-cultured with anotherdonor of PBMCs in the MLR. FIG. 10 depicts a correlation between theamount of CD45+ cells present in amnion-derived cell suspensions andimmunogenicity in MLR in vitro. Table 10 and FIG. 10 show that thesuspensions spiked with higher amounts of live CD45+ cells resulted inhigher immunogenicity as measured by IL-2sR expression on the hPBMCresponder donor.

TABLE 10 % CD45+ recovery experiments. Actual Cell spike (%, suspensionSample based on % Dif- immunogenicity Description % CD45+ 60% CD45+ference (tested in MLR (in % of cell cells cells in from and expressedtypes in the (detected this hPBMC actual as IL-2R in mixture) by FACS)batch) spike pg/mL) 100% amnion 0.65 N/A N/A 20.23 0% PBMC N/A N/A N/A15.6 (negative control) 100% PBMC 61.51 N/A N/A 86.31 (positive control)20% PBMC + 4.26 12 64.5% 24.38 80% Amnion 10% PBMC + 2.24 6 62.7% 21.1790% Amnion 5% PBMC + 1.7 3 43.3% 16.75 95% Amnion 2.5% PBMC + 1.36 1.5Not cal- 15.9  97.5% Amnion culated* 1.25% 1.06 0.75 Not cal- 12.27PBMC + culated* 98.75% Amnion Notes: N/A—not applicable; *Notcalculated - values are close to the method detection limits.

Example 15 Protein Array Analyses

The protein profiles of amniotic and chorionic membranes wereinvestigated using a SearchLight Multiplex chemiluminescence array. Thepresence of proteins in tissue membrane extracts and secreted by tissuesin culture medium was investigated. For comparison, two commerciallyavailable products containing living cells, Apligraf and Dermagraft,were assayed.

Example 15.1 Dermagraft

Dermagraft membrane was thawed and washed according to themanufacturer's instructions. Dermagraft membrane was cut into 7.5 cm²pieces. For tissue lysates, one 7.5 cm² piece of membrane was snapfrozen in liquid nitrogen followed by pulverization using a mortar andpestle. Crushed tissue was transferred to a 1.5 mL microcentrifuge tubeand 500 μL of Lysis buffer (Cell Signaling Technologies, Cat No. 9803)with protease inhibitor (Roche, Cat No. 11836153001) was added andincubated on ice for 30 min with frequent vortexing. The sample was thencentrifuged at 16000 g for 10 min. The supernatant was collected andsent for protein array analysis by Aushon Biosystems. For tissueculture, one 7.5 cm² piece of membrane was plated onto a well of a12-well dish and 2 mL of DMEM+1% HSA+ antibiotic/antimycotic were addedand incubated at 37° C.±2° C. for 3, 7, or 14 days. After incubation,tissue and culture media were transferred to a 15 mL conical tube andcentrifuged at 2000 rpm for 5 min. Culture supernatant was collected andsent for protein array analysis by Aushon Biosystems.

Example 15.2 Apligraf

Apligraf membrane was cut into 7.3 cm² pieces. For tissue lysates, one7.3 cm² piece of membrane was snap frozen in liquid nitrogen followed bypulverization using a mortar and pestle. Crushed tissue was transferredto a 1.5 mL microcentrifuge tube and 500 μL of Lysis buffer (CellSignaling Technologies, Cat No. 9803) with protease inhibitor (Roche,Cat No. 11836153001) was added and incubated on ice for 30 min withfrequent vortexing. The sample was then centrifuged at 16000 g for 10min. The supernatant was collected and sent for protein array analysisby Aushon Biosystems. For tissue culture, one 7.3 cm2 piece of membranewas plated onto a well of a 12-well dish and 2 mL of DMEM+1% HSA+antibiotic/antimycotic were added and incubated at 37° C.±2° C. for 3,7, or 14 days. After incubation, tissue and culture media weretransferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5min. Culture supernatant was collected and sent for protein arrayanalysis by Aushon Biosystems.

Example 15.3 Chorionic Membranes

Chorionic membranes were isolated and packaged at −80° C.±5° C.according to the manufacturing protocols disclosed herein in Example 2.Packaged membranes were then thawed in a 37° C.±2° C. water bath andwashed 3 times with DPBS. Membranes were cut into 8 cm² pieces. Fortissue lysates, one 8 cm² piece of membrane was snap frozen in liquidnitrogen followed by pulverization using a mortar and pestle. Crushedtissue was transferred to a 1.5 mL microcentrifuge tube and 500 μL ofLysis buffer (Cell Signaling Technologies, Cat No. 9803) with proteaseinhibitor (Roche, Cat No. 11836153001) was added and incubated on icefor 30 min with frequent vortexing. Tissue lysate was then centrifugedat 16000 g for 10 min. The supernatant was collected and sent forprotein array analysis by Aushon Biosystems. For tissue culture, one 8cm² piece of membrane was plated onto a well of a 12-well dish and 2 mLof DMEM+1% HSA+ antibiotic/antimycotic were added and incubated at 37°C.±2° C. for 3, 7, or 14 days. After incubation, tissue and culturemedia were transferred to a 15 mL conical tube and centrifuged at 2000rpm for 5 min. Culture supernatant was collected and sent for proteinarray analysis by Aushon Biosystems.

Initial testing consisted of an analysis of 36 proteins that areimportant for wound healing. The list of identified proteins isdescribed in Table 11.

TABLE 11 List of selected proteins for analysis. Protein Group Based onFunctionality Comments Metalloproteases Matrix Metalloproteinase 1Matrix and growth factor (MMP1), MMP2, 3, 7, 8, 9, degradation;facilitate cell 10, 13 migration. MMP Inhibitors Tissue Inhibitors ofMMPs Have angiogenic activity; (TIMP1 and 2) can be placed in the“angiogenic factors” group. Angiogenic Factors Angiotensin-2 (Ang-2);basic Majority of these factors Fibroblast Growth Factor also havegrowth and (bFGF); heparin-bound migration stimulatory Epidermal GrowthFactor activities and can be (HB-EGF); EGF; FGF-7 (also placed in agroup of known as Keratinocyte growth factors. Growth Factor-KGF);Platelet derived Growth Factors (PDGF) AA, AB, and BB; VascularEndothelial Growth Factor (VEGF), VEGF-C and VEGF-D; Neutrophilgelatinase-associated lipocalin (NGAL); Hepatocyte Growth Factor (HGF);Placenta Growth Factor (PIGF); Pigment Epithelium Derived Factor (PEGF);Thrombopoetin (TPO) Protease Inhibitor/ Alpha-2-macroglobulin Inhibitprotease activity; Protein Carrier regulate growth factor activity.Growth Factors See “angiogenic factors” + See “angiogenic factors.”Transforming Growth Factor alpha (TGF-a) Cytokines Adiponectin (Acrp-30)Affect keratinocyte functions. Granulocyte Colony- Protection frominfections. Stimulating Factor (G-CSF) Interleukin1 Receptor Regulateactivity of Antagonist (IL-1RA) inflammatory cytokine IL-1. LeukemiaInhibitory Factor Support angiogenic (LIF) growth factors. ChemokinesSDF-1beta Attracts endothelial and other stem cells from circulation towound site. Regulators of IGF Insulin-like growth factor Regulate IGFactivity. binding protein (IGFBP1, 2, 3)

Example 15.4 Protein Expression in Present Placental Products

Preliminary protein array data analyses showed that the majority ofselected testing factors (see Table 11) were expressed in amnioticmembrane, chorionic membrane, Apligraf, and Dermagraft.

Three proteins were identified as unique for the chorionic membranewhich are undetectable in Apligraf and Dermagraft. These proteins areEGF, IGFBP1, and Adiponectin). FIG. 11 depicts expression of EGF (A),IGFBP1 (B), and Adiponectin (C) in amniotic or chorionic membranes. CM75and CM 78 are placental products of the present invention (e.g.cryopreserved), AM75 and AM78 are cryopreserved amniotic membraneproducts. These proteins are believed by the inventors to facilitate thetherapeutic efficacy of the present placental products for woundhealing.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising a chorionicmembrane containing EGF, IGFBP1, and/or adiponectin.

Example 16 Wound Healing Proteins are Secreted for a Minimum of 14 Days

Placental products of the present invention demonstrate a durableeffect, desirable for wound healing treatments. The extracellular matrixand presence of viable cells within the amniotic membrane described inthis invention allow for a cocktail of proteins that are known to beimportant for wound healing to be present for at least 14 days. Amnioticmembranes were thawed and plated onto tissue culture wells and incubatedat 37° C.±2° C. for 3, 7, and 14 days. At each time point, a sample ofthe culture supernatant was collected and measured through protein arrayanalysis as described in Example 15. Table 12 illustrates the level ofvarious secreted factors in tissue culture supernatants from two donorsof chorionic membranes at 3, 7 and 14 days as measured through proteinarray analysis.

TABLE 12 Levels of proteins secreted in chorion tissue culturesupernatants at different time points (pg/ml). Day 3 Day 7 hACRP30 298.4614.3 hAlpha2Macroglobulin 34,480.5 6,952.5 hANG2 0.0 2.0 hEGF 0.7 0.4hFGF 84.3 13.5 hFibronectin 37,510.9 41,871.4 hHBEGF 102.6 40.0 hHGF1,382.9 1,715.4 hIGFBP1 201.6 201.0 hIGFBP2 62.7 172.9 hIGFBP3 778.1812.4 hIL1ra 30,037.4 556.1 hKGF 4.2 2.4 hMMP1 32,388.5 67,665.6 hMMP104,016.4 4,140.1 hMMP13 13.3 0.0 hMMP2 768.8 1,230.5 hMMP3 1,294.72,646.0 hMMP7 14.7 43.7 hMMP8 95.9 249.4 hMMP9 10,034.6 29,201.5 hNGAL1,968.1 2,608.9 hPDGFAA 18.6 21.8 hPDGFAB 6.2 55.5 hPDGFBB 15.1 5.2hPEDF 9,216.2 576,962.0 hSDF1b 85.9 15.3 hTGFa 0.0 0.0 hTGFb1 377.5410.9 hTGFb2 11.2 20.7 hTIMP1 12,279.0 15,562.7 hTIMP2 216.7 419.6 hTSP1223.1 0.0 hTSP2 42.7 210.7 hVEGF 53.1 45.9 hVEGFC 197.4 182.7

Example 17 Interferon 2α (IFN-2α) and Transforming Growth Factor-β3(TGF-β3)

Placental products described in this invention have been analyzed forthe presence of IFN-2α and TGF-β3. Briefly, after thawing, the membraneswere homogenized and centrifuged at 16,000 g to collect the resultingsupernatants. Supernatants were analyzed on a commercially availableELISA kit from MabTech (IFN-2α) and R&D Systems (TGF-β3).

FIG. 12 shows significant expression of IFN-2α (A) and TGF-β3 (B) incellular chorionic membrane homogenates.

Without being bound by theory, interferon-2α and TGF-β3 may aid in theprevention of scar and contracture formation. IFN-2α may serve a role todecrease collagen and fibronectin synthesis and fibroblast-mediatedwound contracture.

Example 18 Tissue Reparative Proteins in Chorionic Membranes

Chorionic membrane homogenates were analyzed for the presence ofproteins that are important in tissue repair.

Chorionic membranes described in this invention have been analyzed forthe presence of tissue reparative proteins. Briefly, amniotic membraneswere incubated in DMEM+10% FBS for 72 hrs. The membranes were thenhomogenized in a bead homogenizer with the culture media. Thehomogenates were centrifuged, and the supernatants were analyzed oncommercially available ELISA kits from R&D Systems. Significantexpression of BMP-2, BMP-4, PLAB, PIGF, and IGF-1 in several donors ofchorionic membranes.

Without being bound by theory, the inventors believe that efficacy ofthe present placental products for wound repair are due, in part, to therole of BMPs, IGF-1, and PIGF in the development and homeostasis ofvarious tissues by regulating key cellular processes. BMP-2 and BMP-4may stimulate differentiation of MSCs to osteoblasts in addition topromote cell growth; placental BMP or PLAB is a novel member of the BMPfamily that is suggested to mediate embryonic development. Insulin-likegrowth factor 1 (IGF-1) may promotes proliferation and differentiationof osteoprogenitor cells. Placental derived growth factor (PIGF) mayacts as a mitogen for osteoblasts.

Example 19 MMPs and TIMPs

Both MMPs and TIMPs are among the factors that are important for woundhealing. However, expression of these proteins must be highly regulatedand coordinated. Excess of MMPs versus TIMPS is a marker of poor chronicwound healing. We investigated expression of MMPs and TIMPs and itsratio in amniotic membrane and chorionic membrane and compared it to theexpression profile in Apligraf and Dermagraft.

Results showed that all membranes express MMPs and TIMPs; the ratio inthe thawed placental products and amniotic membranes is significantlylower. Therefore, the placental products (optionally including chorionicmembranes) will be more beneficial for wound healing.

Accumulated data indicate that the MMP to TIMP ratio is higher in casesof non-healing wounds. For example, the ratio between MMP-9 and TIMP1 isapproximately 7-10 to one or good healing and 18-20 to one or higher forpoor healing.

As shown in FIG. 14, analysis of the ratio between MMPs and TIMPssecreted by placental tissues, Apligraf, and Dermagraft showed that thechorionic membrane products contain MMPs and TIMPs at an approximateratio of 7, which is favorable for wound healing. In contrast,Dermagraft had a ratio >20, and Apligraf had a ratio >200.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising a chorionicmembrane containing MMP-9 and TIMP1 at a ratio of about 7-10 to one.

Example 20 α2-Macroglobulin

α2-macroglobulin is known as a plasma protein that inactivatesproteinases from all 4 mechanistic classes. Another important functionof this protein is to serve as a reservoir for cytokines and growthfactors, examples of which include TGF, PDGF, and FGF. In the chronicwounds like diabetic ulcers or venous ulcers, the presence of highamount of proteases leads to rapid degradation of growth factors anddelays in wound healing. Thus, the presence of α2-macroglobulin inproducts designed for chronic wound healing will be beneficial. Resultsof the protein array analysis showed that amniotic and chorionicmembranes contain α2-macroglobulin (Table 13).

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising a chorionicmembrane containing α2-macroglobulin.

TABLE 13 Expression of α2-macroglobulin in placental tissue proteinextracts. α2-macroglobulin Sample (pg/mL/cm²) AM75 7 CM75 790 AM78 53042CM78 1014

Example 21 Establishment of bFGF as a Marker for Chorionic TissuePotency

bFGF modulates a variety of cellular processes including angiogenesis,tissue repair, and wound healing (Presta et al., 2005, Reuss et al.,2003, and Su et al., 2008). In wound healing models, bFGF has been shownto increase wound closure and enhance vessel formation at the site ofthe wound (Greenhalgh et al., 1990). Evaluation of proteins derived fromchorionic membranes prepared pursuant to the presently disclosedmanufacturing process revealed that bFGF is one of the major factors inplacental tissue protein extracts (FIG. 15). FIG. 15 depicts expressionof bFGF by amniotic membranes (AM) and chorionic membranes (CM) detectedduring the protein profile evaluation of placental membranes.

The importance of bFGF for wound healing supports selection of bFGF as apotency marker for evaluation of chorionic membrane productsmanufactured for clinical use pursuant to the present disclosure. Acommercially available ELISA kit from R&D Systems was selected forevaluation of its suitability to measure bFGF secreted by placentalmembranes. ELISA method qualification experiments were designedaccording to FDA and ICH guidances for bioanalytical assay validation(Validation of Analytical Procedures: Text and Methodology Q2 (R1),1994; ICH Harmonized Tripartite Guideline and Guidance for IndustryBioanalytical Method Validation, 2001).

The ELISA procedure was performed according to the manufacturer'sinstructions (bFGF ELISA brochure). The evaluation of the kit wasperformed prior to measurement of bFGF in placental tissue samples.Assay performance was assessed by analyzing linearity, range, lower andupper limits of quantitation (LLOQ and ULOQ), precision, and accuracy.Experimental data suggested that the quantitation range of this assaywas 40-1280 pg/mL bFGF. The intra- and inter-assay CVs ranged from 2.42to 6.23% and 0.59 to 7.02%, respectively. Additionally, sample recoveryanalysis demonstrated accuracy within 20%. This assay showed dilutionallinearity and specificity. Ruggedness was demonstrated by assayinsensitivity to variations introduced by different analysts. Theanalytical performance of the bFGF ELISA indicated that this assay wassuitable for the measurement of bFGF secreted by placental membranes.bFGF ELISA parameters are summarized in Table 14.

TABLE 14 Established ELISA parameters for measuring bFGF in placentahomogenates. Calibration Standard 20-1280 pg/mL Range Assay Quantitation40-1280 pg/mL Range LLOQ 40 pg/mL LOD 20 pg/mL ULOQ 1280 pg/mL

bFGF Expression in Chorionic Membranes

Measurement of bFGF in chorionic membrane preparations has proven to beboth reliable and reproducible. The placental tissue homogenates wereprepared using the “bead” method as described above. Also, secretion ofbFGF in tissue culture media was evaluated. Measurement of bFGF inmultiple donors showed that this method of quantification was a valuablemeans of evaluating potency the presently disclosed tissue productsprepared for use in a clinical setting. FIG. 16 shows representativeexpression of bFGF in chorionic tissue samples derived from two separateplacenta donors. Results have been reproduced in multiple tissuepreparations.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising a chorionicmembrane containing bFGF.

Example 22 Placental Tissues Enhance Cell Migration and Wound Healing

The process of wound healing is highly complex and involves a series ofstructured events controlled by growth factors (Goldman, 2004). Theseevents include increased vascularization, infiltration by inflammatoryimmune cells, and increases in cell proliferation. The beginning stagesof wound healing revolve around the ability of individual cells topolarize towards the wound and migrate into the wounded area in order toclose the wound area and rebuild the surrounding tissue. Upon properstimulation, several different types of cells including epithelial,endothelial, mesenchymal, and fibroblastic cells are implicated in thewound healing process (Pastar et al, 2008 and Bannasch et al., 2000).Specifically, they proliferate and migrate into the wound area topromote healing. Therefore, experiments were conducted to determine iffactors secreted from amniotic and chorionic membranes produced pursuantto the present disclosure promote vrII migration and wound fieldclosure. To accomplish this, a commercially available wound healingassay (Cell Biolabs) and a highly accepted human microvascularendothelial cell line (HMVEC, Lonza Inc.) were utilized. Resultsindicated that cell migration was enhanced by treatment with conditionedmedia from the placental membranes.

In Vitro Cell Migration

Human microvascular endothelial cells (HMVECs) were grown under normalcell culture conditions in defined complete media (Lonza Inc.). Toassess migration and wound field closure, a commercially available woundhealing assay was used (Cell Biolab). The assay principle is outlined inFIG. 17.

FIG. 17 depicts the Cell Biolabs 24-well Cytoselect wound healing assay.(Figure reproduced from Cell Biolabs).

Cells were collected via trypsinization, pelleted, and counted beforebeing resuspended in complete media at a density of 2×10⁵ cells/mL. 250μL (5×10⁴ cells) of cell suspension was then pipetted into each side ofa well containing a wound healing insert (Cytoselect 24-well WoundHealing Assay Plate, Cell Biolabs). The cells were grown for 24 hours incomplete media. After 24 hours, the wound inserts were removed. At thesame time, complete media was removed and replaced with experimentalmedia. Complete media and basal media were used as positive and negativecontrols, respectively. To generate experimental media, placentalmembranes were incubated for 3 days in DMEM with 1% human serum albumin(HSA) in a tissue culture incubator. The resulting tissue and media werethen placed in eppendorf tubes and spun at high speed in amicrocentrifuge. The supernatants were collected and stored at −80°C.±5° C. until use. For migration and wound healing studies, conditionedmedia from placental membranes was diluted 1:20 in basal media beforebeing added to experimental wells. After 18 hours, the media wasremoved, and the cells were fixed for 20 min in 4% paraformaldehyde andstained with crystal violet. The wound field in each well was thenphotographed. Wound healing was determined by the amount of wound fieldstill visible at the end of the experiment when compared to controlpictures taken before conditioned media was added to the wells.

Placental Membrane Conditioned Media Supports Cell Migration and WoundField Closure

Conditioned media from amniotic and chorionic membranes was used toassess the potential for these membranes to promote cell migration andwound field closure. Conditioned media from placental chorionicmembranes supported migration of cells into the experimental woundfield. FIG. 18 depicts representative images of HMVECs treated with 5%conditioned media from amniotic, chorionic, or a combination ofamniotic/chorionic tissue as well as positive and negative controls.Wound field is 0.9 mm in width.

The ability of factors from placental membranes produced pursuant to thepresent disclosure to promote HMVEC migration indicated that thesetissues have the ability to enhance wound healing. Additionally, basedon the insight of the inventors, it has been surprisingly discoveredthat these tissues also enhance revascularization since the HMVEC cellline is derived from vascular endothelial cells.

These data demonstrate that placental products of the present inventionproduce unexpectedly superior levels of factors that promote woundhealing therapies.

Example 23 Analysis of Factors in Exemplary Placental Tissue Products

Table 15 depicts the biochemical profile of examplary placental productsof the invention (results adjusted per cm² after subtraction of thenegative background).

TABLE 15 Factors in placental tissue product (pg/cm²) Units ApligrafDermagraft AM75 CM75 AM78 CM78 hMMP1 pg/ml/cm² 1964945.37 14818.202821.85 3531.81 117326.89 95.46 hMMP7 pg/ml/cm² 911.54 0.00 0.00 0.003.96 0.00 hMMP10 pg/ml/cm² 0.00 0.00 113.94 0.00 0.00 0.00 hMMP13pg/ml/cm² 21.61 0.00 0.00 0.00 0.71 0.00 hMMP3 pg/ml/cm² 208281.70180721.52 170.26 161.52 8325.17 0.00 hMMP9 pg/ml/cm² 8872.28 19321.39214.78 1455.11 630.56 57.59 hMMP2 pg/ml/cm² 153341.77 19712.21 287.1437.93 3823.38 24.44 hMMP8 pg/ml/cm² 36.92 12.19 0.00 0.00 0.00 0.00hTIMP1 pg/ml/cm² 2487.18 10909.84 569.23 883.05 28743.48 97.94 hTIMP2pg/ml/cm² 7285.53 1796.56 89.29 13.72 424.06 4.83 MMP/TIMP 239.26 19.726.81 6.26 4.50 2.62

Example 24 Factors in Exemplary Placental Products as Measured ThroughProtein Array Analysis by Aushon Biosystems

Table 16 depicts the biochemical profile of the lysates of examplaryplacental products of the invention (results adjusted per cm² aftersubtraction of the negative background).

TABLE 16 AM75 lysate AM78 lysate CM75 lysate CM78 lysate pg/ml pg/mlpg/ml pg/ml hACRP30 50.8 1154.6 1213.7 225.3 hAlpha2- 1910.6 426191.68174.4 9968.6 Macroglobulin hEGF 127.3 361.4 0.0 0.8 hbFGF 119.1 821.5375.0 351.3 hGCSF 0.7 3.2 1.2 0.7 hHBEGF 127.5 168.0 15.4 84.5 hHGF3943.7 15060.0 29979.6 50392.8 hIGFBP1 5065.0 9456.6 934.0 1443.6hIGFBP2 12460.8 5569.7 135.9 134.6 hIGFBP3 50115.7 41551.4 4571.511970.2 hIL1ra 3881.0 32296.9 5168.2 525.5 hKGF 1.4 8.8 3.1 1.5 hLIF 0.04.2 0.0 0.0 hMMP1 9144.1 20641.2 2882.9 6582.3 hMMP10 0.0 15.5 79.3 87.5hMMP2 2067.3 4061.9 949.5 748.8 hMMP3 0.0 36.2 0.0 0.0 hMMP7 5.1 11.44.5 9.1 hMMP8 0.0 0.0 0.0 0.0 hMMP9 92.2 2878.1 2676.2 1259.3 hNGAL6900.1 6175.9 938.5 229.7 hPDGFAA 0.0 12.5 39.8 35.2 hPDGFAB 11.2 31.314.4 14.0 hPDGFbb 4.6 13.4 4.0 1.3 hPEDF 0.0 652.6 0.0 0.0 hTIMP1 7958.135955.6 50712.3 17419.9 hTIMP2 3821.8 7443.2 640.7 780.0 hVEGF 3.3 11.8125.2 8.4 hVEGFC 46.5 150.0 123.5 51.7 hVEGFD 25.7 31.0 15.0 20.4

What is claimed is:
 1. A cryopreserved chorionic membrane comprisingviable cells native to the chorionic membrane; wherein the cryopreservedchorionic membrane is immunocompatible; wherein at least about 70% ofthe cells are viable; wherein the viable cells comprise at least twocell types consisting of mesenchymal stem cells (MSCs) and fibroblasts;and wherein the cryopreserved chorionic membrane comprises CD14+macrophages in an amount of less than about 5%.
 2. The cryopreservedchorionic membrane of claim 1, wherein the cryopreserved chorionicmembrane comprises one or more therapeutic factors native to thechorionic membrane.
 3. The cryopreserved chorionic membrane of claim 1,wherein the cryopreserved chorionic membrane comprises extracellularmatrix that is native to the chorionic membrane.
 4. The cryopreservedchorionic membrane of claim 1, wherein the chorionic membrane is madeimmunocompatible by removal of all or a portion of a trophoblast layer.5. The cryopreserved chorionic membrane of claim 1, wherein thechorionic membrane exhibits a substantial decrease in TNF-α releaseafter lipopolysaccharide (LPS) stimulation.
 6. The cryopreservedchorionic membrane of claim 5, wherein the chorionic membrane releasesless than 420 pg/ml of TNF-a.
 7. The cryopreserved chorionic membrane ofclaim 1, wherein the chorionic membrane is substantially free ofimmunogenic maternal cells.
 8. The cryopreserved chorionic membrane ofclaim 7, wherein the maternal cells are maternal dendritic cells,maternal leukocytes, or combinations thereof.
 9. The cryopreservedchorionic membrane of claim 1, wherein the viable cells comprise stromalcells.
 10. The cryopreserved chorionic membrane of claim 9, wherein thestromal cells are present in an amount of about 5,000 per cm² to about50,000 per cm² of the chorionic membrane.
 11. The cryopreservedchorionic membrane of claim 1, further comprising at least onecell-permeating cryopreservative, at least one non-cell-permeatingcryopreservative, derivatives thereof, or a combination thereof.
 12. Thecryopreserved chorionic membrane of claim 11, wherein thecell-permeating cryopreservative, the non-cell-permeatingcryopreservative, derivative thereof, or combination thereof is presentin an amount of about 5% to about 20% by volume.
 13. The cryopreservedchorionic membrane of claim 12, wherein the cell-permeatingcryopreservative is DMSO.
 14. The cryopreserved chorionic membrane ofclaim 1, wherein the chorionic membrane comprises fibroblasts present inan amount of about 50% to about 90% of the total cells, and wherein atleast about 40% of the fibroblasts are viable after one freeze-thawcycle.
 15. The cryopreserved chorionic membrane of claim 1, wherein thechorionic membrane comprises mesenchymal stem cells in an amount ofabout 5% to about 30% and wherein at least 40% of the MSCs are viableafter one freeze-thaw cycle.
 16. The cryopreserved chorionic membrane ofclaim 1, wherein the chorionic membrane comprises a whole or partialbasement membrane, a whole or partial reticular layer, or a whole orportion combination thereof.
 17. The cryopreserved chorionic membrane ofclaim 1, wherein the chorionic membrane is associated with anitrocellulose substrate or support.
 18. A membrane comprisingcryopreserved chorionic membrane, wherein the cryopreserved chorionicmembrane comprises: a) a chorionic membrane; b) viable cells, whereinsaid viable cells are native to the chorionic membrane and greater than70% of said cells are viable, wherein the viable cells comprise at leasttwo cell types consisting of MSCs and fibroblasts; c) one or moretherapeutic factors that is native to the chorionic membrane; d)extracellular matrix that is native to the chorionic membrane; and e) anamount of less than about 5% of CD14+ macrophages.
 19. The membrane ofclaim 18, further comprising a cryopreserved amniotic membrane whereinthe amniotic membrane comprises a layer of amniotic epithelial cells.20. The membrane of claim 19, wherein the cryopreserved chorionicmembrane is substantially depleted of placental trophoblasts.
 21. Amethod of treating an injury comprising administering to the injury thecryopreserved chorionic membrane of claim 1 when thawed.
 22. The methodof claim 21, wherein the injury is a wound.
 23. The method of claim 22,wherein the wound is a laceration, a scrape, a thermal or chemical burn,an incision, a puncture, a wound caused by a projectile, an epidermalwound, a skin wound, a chronic wound, an acute wound, an external wound,an internal wound, a congenital wound, an ulcer, a pressure ulcer, or acombination thereof.
 24. The method of claim 21, wherein the injurycomprises a wound or injury associated with a surgical procedure. 25.The method of claim 24, wherein the surgical procedure is selected fromthe group consisting of a tendon surgery, a ligament surgery, a bonesurgery, a spinal surgery, a laminectomy, a knee surgery, a shouldersurgery, and child delivery.